外源性PTH1-34可缓解FGFR3功能增强型点突变所致软骨发育障碍及骨折愈合延迟
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摘要
FGFR3的十多种功能增强型(Gain-of-function)点突变可引起多种人类软骨发育障碍性侏儒,包括软骨发育不全(Achondroplasia, ACH)、季肋发育不全(Hypochondroplasia, HCH)、致死性骨发育不全(Thanatophoric Dysplasia, TD)等。95%以上的软骨发育不全由FGFR3的功能增强型点突变引起。软骨发育不全是一种常染色体显性遗传病,是人类侏儒最常见的类型。软骨发育不全主要影响经软骨内成骨(Endochondral Ossification)过程形成的四肢骨等长骨(Long Bone)。这些患者除可通过外科手术进行部分延长肢体外,目前尚无有效的治疗方法。
Chen等利用基因敲入技术建立了模拟人ACH的FGFR3G369C点突变小鼠模型,个体明显短小,头颅短圆,长骨生长板组织形态结构异常,软骨细胞增殖、分化能力减弱;Deng等发现,FGFR3基因敲除小鼠(FGFR3-/-)有长骨过度生长、生长板增殖软骨细胞带和肥大软骨细胞带变宽、软骨细胞增殖活性增加。这些结果提示FGFR3是软骨发育的负性调节分子,影响软骨细胞的增殖活性和分化。
Chen等发现,FGFR3信号通过STATs和Ihh抑制软骨细胞增殖,FGFR3信号可能与PTHrP/IHH信号相互独立抑制软骨细胞分化。Yamanaka等发现表达FGFR3ACH(FGFR3 G380R)和TDII(FGFR3 K650E)突变的ATDC5细胞凋亡增加,给予PTHrP处理后可缓解。Ueda等发现,PTH1-34抑制软骨细胞分化和凋亡,增加骨长度,可以一定程度上缓解FGFR3突变对长骨生长迟缓带来的影响。PTH1-34可能一定程度上缓解FGFR3功能增强对细胞与组织带来的生长受损。那么PTH1-34是否能在动物水平上缓解FGFR3功能增强所引起的软骨发育不全,有待于进一步研究。
FGFR3除影响骨骼发育外,还参与骨折修复的调节。本实验室Su等发现FGFR3在骨折愈合过程中通过抑制软骨形成、分化以及矿化软骨基质降解导致骨折愈合延迟。如何缓解FGFR3激活引起的骨骼发育异常及骨折修复延迟有重要的临床意义。
重组人PTH1-34氨基酸片段的多肽类激素药物特立帕肽(teriparatide)是得到美国食品与药物管理局(FDA)批准的用于临床治疗绝经后妇女的骨质疏松的药物,其机制与其促进成骨有关。此外,一些工作观察了其对骨折愈合的影响。Alkhiary和Nakazawa研究发现PTH1-34可能通过增加软骨形成来刺激软骨内成骨。PTH1-34是否可缓解ACH小鼠由于软骨形成分化障碍所引起的骨折愈合延迟还有待于研究。
据此,本课题利用野生型和FGFR3G369C/+(ACH)小鼠,间断给予外源性PTH处理,通过分析各组小鼠的骨骼表型,观察外源性PTH对FGFR3功能增强型点突变所致的软骨发育障碍的影响。此外,我们还初步观察了外源性PTH对FGFR3功能增强型点突变所致的骨折愈合延迟的影响。
主要实验方法:
第一部分:外源性PTH1-34对FGFR3G369C/+小鼠骨骼生长抑制的影响
采用X线摄影、全骨架染色和头颅局部摄影,观察小鼠大体形态及颅底软骨联结的变化情况,测定小鼠生长过程中体重、体长等的变化。
1.动物模型:采用新出生生当日的WT及ACH小鼠,分别随机分为给药组与对照组,给药组给予80μg/(kg·d) PTH1-34皮下注射,对照组给予等量注射用水直至观察期结束;
2.制备了不同年龄不同组别小鼠的组织切片,通过HE、藏红固绿染色观察生长板形态和第二骨化中心的发育情况;通过BrdU掺入后免疫组化检测,观察生长板软骨细胞增殖情况;
3.测量小鼠生长板肥大带的宽度,采用定量PCR检测软骨组织COL2A1、COL10A1和PTHrP的表达情况;
4.建立胎鼠胫骨及跖骨体外培养模型;采用PTH1-34间断处理培养的野生、突变(ACH)小鼠趾骨,观测其对跖骨生长的影响;
5.PTH处理培养的原代软骨细胞,观察其增殖分化的改变以及对FGFR3表达水平的影响。
第二部分:外源性PTH1-34对FGFR3G369C/+小鼠骨折愈合延迟的影响
1.制作小鼠胫骨近端稳定骨折模型,各基因型分别随机分为给药组与对照组,术后当日起给药组给予80μg/(kg·d) PTH1-34皮下注射,对照组给予等量注射用水直至观察期结束;
2.利用X线摄片和藏红固绿染色观察骨折愈合情况;
3.检测小鼠血清钙、磷含量;
4.检测骨痂中软骨细胞分化相关基因COL2A1、COL10A1、PTHrP和成骨相关的基因cbfa-1和OCmRNA变化观察软骨分化情况。
主要实验结果:
一、外源性PTH1-34可部分缓解FGFR3功能增强所致的骨骼生长抑制
(一)对FGFR3功能增强型点突变小鼠一般生长情况的影响
在观测期1 w-8 w内,给予PTH1-34的ACH小鼠体重、鼻-肛门长度增长较对照ACH小鼠明显;颅底软骨连接提前闭合有所改善;枕骨大孔狭窄无明显改善。
(二)对FGFR3功能增强型点突变小鼠软骨内成骨的影响
1.BrdU掺入检测后发现,给予PTH1-34的ACH小鼠软骨细胞增殖指数较对照ACH小鼠高;P16给予PTH1-34的ACH小鼠生长板软骨细胞增殖带较ACH宽;原代软骨细胞培养细胞计数与MTT检测发现给予PTH1-34处理的ACH小鼠软骨细胞增殖活性增强。
2.观察出生14 d小鼠的生长板,发现给予PTH1-34的ACH小鼠的第二骨化中心较ACH对照小鼠的提前出现,P7、P10,给予PTH1-34的ACH小鼠生长板肥大带明显较ACH对照小鼠增宽;荧光定量PCR提示:给予PTH1-34的ACH小鼠生长板软骨及原代细胞的COL10A1 mRNA表达水平较ACH-小鼠高。
(四)胚胎跖骨培养
建立了跖骨体外培养7 d的模型,ACH胎鼠(E16.5)跖骨在给予PTH1-34间断处理后提示:处理组跖骨生长率明显较未处理组高,主要增长的是软骨区域,提示:间断PTH1-34处理可促进软骨生长。
(五)可能机制的研究
外源性PTH1-34处理原代软骨细胞后FGFR3的mRNA及蛋白质水平均降低。
二、外源性PTH1-34可部分缓解FGFR3功能增强所致的骨折愈合延迟
1.X光摄片提示给药组骨痂密度和骨连接程度较对照组高:骨折14天,ACH对照组小鼠骨痂较处理组小,而且钙化密度较低;骨折28天,ACH对照小鼠骨折线隐约可见,而ACH给药组小鼠骨折基本愈合;
2.藏红固绿染色提示:骨折7天,给予PTH1-34的ACH小鼠骨痂中出现大量软骨,并且部分已进入肥大期;骨折14天,PTH1-34给药组ACH小鼠的软骨痂体积小于对照组;骨折21天,ACH对照组小鼠的骨痂中仍可见残留软骨组织,而ACH给药组小鼠中未见;
3.给予外源性PTH1-34对血清钙磷无明显影响,定量PCR提示,骨折早期,ACH给药组骨痂中的COL10A1 mRNA表达增高,PCNA mRNA表达增高;骨折中期,ACH给药组骨痂中的OC mRNA表达增高。
主要结论:
一、外源性PTH1-34可部分缓解FGFR3功能增强所致软骨发育不全
1.间断给予PTH1-34的ACH小鼠大体表型较ACH对照小鼠有所缓解;
2.间断给予PTH1-34可使ACH软骨细胞增殖活性较ACH对照小鼠增高;
3.间断给予PTH1-34可使ACH生长板软骨细胞分化较ACH对照小鼠有所改善;
4.间断给予PTH1-34处理可以促进小鼠跖骨生长;
5.间断给予PTH1-34处理促进软骨细胞的增殖分化可能与FGFR3的表达降低有关。
二、外源性PTH1-34可部分缓解FGFR3功能增强所致骨折愈合延迟
外源性PTH1-34可通过促进小鼠骨折早期软骨痂的形成及中后期软骨痂的改建,加速软骨向骨转化来改善ACH小鼠的骨折愈合延迟。
A dozen or more activated mutations in the coding sequence of the FGFR3 gene can cause a variety of human dwarfism with developmental disorders,such as achondroplasia (Ach) ,hypochondroplasia (HCH), thanatophoric dysplasia (TD), and some other related disorders.ACH is the most common form of dwarfism and a kind of autosomal dominant inheritable disease.The characteristic phenotypes of ACH include rhizomeric short limbs, macrocephaly, and lumbarlordosis.There is no effective therapy for ACH so far other than bone lengthening.
Chen et al made a knock-in mouse model (FGFR3G369C) mimicing achondroplasia and found that this model had the bone phenotypes, such as small habius, short and round skull, structural abnormality of growth plate, disturbed proliferation and differentiation of growth plate chondrocytes and so on. Deng et al identified the role of FGFR3 in endochondral ossification by disrupting the murine FGFR3 gene.FGFR3 knock-out mice (FGFR3-/-) exhibited enhanced and prolonged endochondral bone growth accompanied by expansion of proliferating and hypertrophic chondrocytes within the cartilaginous growth plate. FGFR3 appeared to regulate endochondral ossification by an essentially negative mechanism .
Chen et al reported that FGF-FGFR3 signaling targets STATs and IHH to inhibit chondrocyte proliferation , while FGF-FGFR3 singnaling and PTHrP/IHH singaling may inhibit chondrocyte differentiation independently. It has been found that both Ach-(FGFR3 G380R) and TDII-type(FGFR3 K650E) mutant FGFR3 induce apoptotic changes and marked decrease of PTHrP expression in ATDC5 cells, a mouse chondrogenic cell line.PTHrP treatment can block the apoptotic response induced by FGFR3. Bone culture study has suggested that PTH1-34 inhibited differentiation and apoptosis of chondrocytes and improved bone growth,which indicates that PTH1-34 may be used as a potential therapeutic agent for achondroplasia.Whether or not the systemic administration of PTH1-34 is a pharmacological therapy for ACH needs to be further studied.
As a regulator of bone growth, FGFR3 is also involved in the regulation of fracture repair.Su et al reported that gain-of-function mutation of FGFR3 resulted in delayed fracture healing by inhibiting chondrocyte differentiation and bone resorption. Bone lengthening,a frequently performed surgery in patients with ACH, also involves fracture healing process. Thus ,searching for the biological measure to improve the bone healing in patients with ACH is very important.
Recombined human PTH1-34(Teriparatide) is the FDA-approved drug to treat osteoporosis in humans by promoting bone formation. There have been a variety of studies on the role of PTH1-34 in fracture healing,which demonstrated that PTH1-34 preferentially enhanced chondrocyte recruitment the rate of chondrocyte maturation to stimulate endochondral ossification. Whether or not PTH1-34 is an effective biological therapeutic measure for the delayed fracture repair in ACH needs further study.
In this study, we used mouse model mimicking human achondroplasia caused by gain-of-function mutation of FGFR3 (FGFR3G369C/+ mice) to explore the effect of PTH1-34 on the achondroplasia and delayed fracture healing resulting from ACH.
Methods
Part I Study of the effects of exogenous PTH1-34 on the bone development retardation of FGFR3G369C/+ mice
1. Animal model: New born wild-type(WT) and ACH mice were randomly divided into treatment group and control group. The mice in treatment group were given PTH1-34 80μg/(kg·d) subcutaneously until the end of the observation period while the control mice were given sterile water.
2. The body weight as well as length of naso-anal, tibia and femur were measured, whole skeleton staining and skull photography were performed. The cranial synchondrosis and foramen magnum of mice were observed.
3. Histological sections of mice at different ages were stained with HE and safranine-fast green . Chondrocyte proliferation in the growth plate was investigated by BrdU labeling assay.
4. The lengths of hypertrophic chondrocyte zone were measured. The expression of some marker genes during chondrocyte differentiation including typeⅡcollagen, type X collagen and PTHrP were detected by real-time PCR.
5. The bone culture system of metatarsal in vitro was established, metatarsal growth rates with or without PTH1-34 were determined and compared.
6. Intermittent PTH1-34 treatment were performed in primary cultured chondrocytes. Cell proliferation was detected using an in vitro MTT colorimetric assay and cytometry. The expression levels of FGFR3 were detected by real-time PCR and western blotting.
Part II Study of the effects of exogenous PTH1-34 on the delayed fracture healing of FGFR3G369C/+ mice
1. Closed fracture of proximal tibia was created and stabilized with an intramedullary pin in 7-8-week-old mice. The mice were randomly divided into treatment group and control group. The mice in treatment group were given PTH1-34 80μg/(kg·d) subcutaneously until the end of the observation period while the control mice were given sterile water.
2. Callus tissues were analyzed at 1-4 weeks post-fracture by radiography and histology.
3. The concentration of serum calcium and phosphorus were measured.
4. RNA was isolated from callus tissues, and the expression levels of bone formation-related genes were evaluated by real-time PCR.
Results
Part I Exogenous PTH can rescue the retarded bone development in ACH mice caused by carrying gain-of-function mutation of FGFR3
1. The naso-anal lengths of ACH mice treated by PTH1-34 at 4th week were significant longer than that in ACH control mice.The cranial synchondrosis time of ACH mice treated by PTH1-34 was delayed compared with that in ACH control mice.The size of foramen magnum had no significant difference.
2. The chondrocyte proliferative index measured by BrdU labeling assay indicated that the chondrocyte proliferative index in ACH mice treated by PTH1-34 was significantly higher than that in ACH control mice. The cell culture study confirmed this result.
3. On postnatal 7 d and 10 d, the hypertrophic zone of chondrocytes in growth plate of PTH1-34-treated ACH mice was wider than that in ACH control mice, and the secondary ossification center was observed advanced in PTH1-34 treated ACH mice..Real-time PCR measurement of both tissues and cells had comfirmed that.
4. Analysis suggested that the tibial growth rate of ACH mice was significantly slower than that in littermate WT control. PTH1-34-treatment can improve the growth suppression in ACH mice.
5. The expression of FGFR3 was decreased by PTH1-34 treatment.
Part II Exogenous PTH1-34 can rescue the delayed fracture healing caused by gain-of-function mutation of FGFR3 in mice
1. At 7 days post-fracture, cartilaginous callus areas were increased in PTH1-34-treated ACH mice compared with those of control mice. In contrast, at 14 days post-fracture, the remnant cartilaginous callus areas were smaller in PTH1-34-treated mice than those in control mice. There were remnant cartilaginous callus in ACH control mice while little remnant cartilaginous callus was observed at 21 days after fracture. The remnant fibrous bone areas in marrow cavity were also reduced in PTH1-34-treated mice compared to control mice at 28 days after fracture.
2. In the early stages of fracture (7 days), compared with the control group, the PCNA and COL10A1 mRNAs expression were obviously increased in PTH1-34 treatment group. In the middle stages of fracture(14 days), compared with the control group, the OC mRNA expression level were significantly increased in PTH1-34 treatment group
Conclusions:
1. The gross phenotypes of ACH mice treated with PTH1-34 including body weight and body length were increased compared with that in ACH control mice.
2. The proliferative and differentiation activity of chondrocytes in growth plates of ACH mice were inhanced by intermittent PTH1-34 treatment.
3. Results of PTH1-34 treatment on metatarsals suggested that PTH1-34 could promote the bone elongation of ACH mice.
4. The rescuing effect of PTH1-34 on bone retardation of ACH mice may be related to down regulation of FGFR3 expression.
5. Exogenous PTH1-34 promoted bone fracture healing in terms of increasing callus areas, endochondral ossification.
Chen等利用基因敲入技术建立了模拟人ACH的FGFR3G369C点突变小鼠模型,个体明显短小,头颅短圆,长骨生长板组织形态结构异常,软骨细胞增殖、分化能力减弱;Deng等发现,FGFR3基因敲除小鼠(FGFR3-/-)有长骨过度生长、生长板增殖软骨细胞带和肥大软骨细胞带变宽、软骨细胞增殖活性增加。这些结果提示FGFR3是软骨发育的负性调节分子,影响软骨细胞的增殖活性和分化。
Chen等发现,FGFR3信号通过STATs和Ihh抑制软骨细胞增殖,FGFR3信号可能与PTHrP/IHH信号相互独立抑制软骨细胞分化。Yamanaka等发现表达FGFR3ACH(FGFR3 G380R)和TDII(FGFR3 K650E)突变的ATDC5细胞凋亡增加,给予PTHrP处理后可缓解。Ueda等发现,PTH1-34抑制软骨细胞分化和凋亡,增加骨长度,可以一定程度上缓解FGFR3突变对长骨生长迟缓带来的影响。PTH1-34可能一定程度上缓解FGFR3功能增强对细胞与组织带来的生长受损。那么PTH1-34是否能在动物水平上缓解FGFR3功能增强所引起的软骨发育不全,有待于进一步研究。
FGFR3除影响骨骼发育外,还参与骨折修复的调节。本实验室Su等发现FGFR3在骨折愈合过程中通过抑制软骨形成、分化以及矿化软骨基质降解导致骨折愈合延迟。如何缓解FGFR3激活引起的骨骼发育异常及骨折修复延迟有重要的临床意义。
重组人PTH1-34氨基酸片段的多肽类激素药物特立帕肽(teriparatide)是得到美国食品与药物管理局(FDA)批准的用于临床治疗绝经后妇女的骨质疏松的药物,其机制与其促进成骨有关。此外,一些工作观察了其对骨折愈合的影响。Alkhiary和Nakazawa研究发现PTH1-34可能通过增加软骨形成来刺激软骨内成骨。PTH1-34是否可缓解ACH小鼠由于软骨形成分化障碍所引起的骨折愈合延迟还有待于研究。
据此,本课题利用野生型和FGFR3G369C/+(ACH)小鼠,间断给予外源性PTH处理,通过分析各组小鼠的骨骼表型,观察外源性PTH对FGFR3功能增强型点突变所致的软骨发育障碍的影响。此外,我们还初步观察了外源性PTH对FGFR3功能增强型点突变所致的骨折愈合延迟的影响。
主要实验方法:
第一部分:外源性PTH1-34对FGFR3G369C/+小鼠骨骼生长抑制的影响
采用X线摄影、全骨架染色和头颅局部摄影,观察小鼠大体形态及颅底软骨联结的变化情况,测定小鼠生长过程中体重、体长等的变化。
1.动物模型:采用新出生生当日的WT及ACH小鼠,分别随机分为给药组与对照组,给药组给予80μg/(kg·d) PTH1-34皮下注射,对照组给予等量注射用水直至观察期结束;
2.制备了不同年龄不同组别小鼠的组织切片,通过HE、藏红固绿染色观察生长板形态和第二骨化中心的发育情况;通过BrdU掺入后免疫组化检测,观察生长板软骨细胞增殖情况;
3.测量小鼠生长板肥大带的宽度,采用定量PCR检测软骨组织COL2A1、COL10A1和PTHrP的表达情况;
4.建立胎鼠胫骨及跖骨体外培养模型;采用PTH1-34间断处理培养的野生、突变(ACH)小鼠趾骨,观测其对跖骨生长的影响;
5.PTH处理培养的原代软骨细胞,观察其增殖分化的改变以及对FGFR3表达水平的影响。
第二部分:外源性PTH1-34对FGFR3G369C/+小鼠骨折愈合延迟的影响
1.制作小鼠胫骨近端稳定骨折模型,各基因型分别随机分为给药组与对照组,术后当日起给药组给予80μg/(kg·d) PTH1-34皮下注射,对照组给予等量注射用水直至观察期结束;
2.利用X线摄片和藏红固绿染色观察骨折愈合情况;
3.检测小鼠血清钙、磷含量;
4.检测骨痂中软骨细胞分化相关基因COL2A1、COL10A1、PTHrP和成骨相关的基因cbfa-1和OCmRNA变化观察软骨分化情况。
主要实验结果:
一、外源性PTH1-34可部分缓解FGFR3功能增强所致的骨骼生长抑制
(一)对FGFR3功能增强型点突变小鼠一般生长情况的影响
在观测期1 w-8 w内,给予PTH1-34的ACH小鼠体重、鼻-肛门长度增长较对照ACH小鼠明显;颅底软骨连接提前闭合有所改善;枕骨大孔狭窄无明显改善。
(二)对FGFR3功能增强型点突变小鼠软骨内成骨的影响
1.BrdU掺入检测后发现,给予PTH1-34的ACH小鼠软骨细胞增殖指数较对照ACH小鼠高;P16给予PTH1-34的ACH小鼠生长板软骨细胞增殖带较ACH宽;原代软骨细胞培养细胞计数与MTT检测发现给予PTH1-34处理的ACH小鼠软骨细胞增殖活性增强。
2.观察出生14 d小鼠的生长板,发现给予PTH1-34的ACH小鼠的第二骨化中心较ACH对照小鼠的提前出现,P7、P10,给予PTH1-34的ACH小鼠生长板肥大带明显较ACH对照小鼠增宽;荧光定量PCR提示:给予PTH1-34的ACH小鼠生长板软骨及原代细胞的COL10A1 mRNA表达水平较ACH-小鼠高。
(四)胚胎跖骨培养
建立了跖骨体外培养7 d的模型,ACH胎鼠(E16.5)跖骨在给予PTH1-34间断处理后提示:处理组跖骨生长率明显较未处理组高,主要增长的是软骨区域,提示:间断PTH1-34处理可促进软骨生长。
(五)可能机制的研究
外源性PTH1-34处理原代软骨细胞后FGFR3的mRNA及蛋白质水平均降低。
二、外源性PTH1-34可部分缓解FGFR3功能增强所致的骨折愈合延迟
1.X光摄片提示给药组骨痂密度和骨连接程度较对照组高:骨折14天,ACH对照组小鼠骨痂较处理组小,而且钙化密度较低;骨折28天,ACH对照小鼠骨折线隐约可见,而ACH给药组小鼠骨折基本愈合;
2.藏红固绿染色提示:骨折7天,给予PTH1-34的ACH小鼠骨痂中出现大量软骨,并且部分已进入肥大期;骨折14天,PTH1-34给药组ACH小鼠的软骨痂体积小于对照组;骨折21天,ACH对照组小鼠的骨痂中仍可见残留软骨组织,而ACH给药组小鼠中未见;
3.给予外源性PTH1-34对血清钙磷无明显影响,定量PCR提示,骨折早期,ACH给药组骨痂中的COL10A1 mRNA表达增高,PCNA mRNA表达增高;骨折中期,ACH给药组骨痂中的OC mRNA表达增高。
主要结论:
一、外源性PTH1-34可部分缓解FGFR3功能增强所致软骨发育不全
1.间断给予PTH1-34的ACH小鼠大体表型较ACH对照小鼠有所缓解;
2.间断给予PTH1-34可使ACH软骨细胞增殖活性较ACH对照小鼠增高;
3.间断给予PTH1-34可使ACH生长板软骨细胞分化较ACH对照小鼠有所改善;
4.间断给予PTH1-34处理可以促进小鼠跖骨生长;
5.间断给予PTH1-34处理促进软骨细胞的增殖分化可能与FGFR3的表达降低有关。
二、外源性PTH1-34可部分缓解FGFR3功能增强所致骨折愈合延迟
外源性PTH1-34可通过促进小鼠骨折早期软骨痂的形成及中后期软骨痂的改建,加速软骨向骨转化来改善ACH小鼠的骨折愈合延迟。
A dozen or more activated mutations in the coding sequence of the FGFR3 gene can cause a variety of human dwarfism with developmental disorders,such as achondroplasia (Ach) ,hypochondroplasia (HCH), thanatophoric dysplasia (TD), and some other related disorders.ACH is the most common form of dwarfism and a kind of autosomal dominant inheritable disease.The characteristic phenotypes of ACH include rhizomeric short limbs, macrocephaly, and lumbarlordosis.There is no effective therapy for ACH so far other than bone lengthening.
Chen et al made a knock-in mouse model (FGFR3G369C) mimicing achondroplasia and found that this model had the bone phenotypes, such as small habius, short and round skull, structural abnormality of growth plate, disturbed proliferation and differentiation of growth plate chondrocytes and so on. Deng et al identified the role of FGFR3 in endochondral ossification by disrupting the murine FGFR3 gene.FGFR3 knock-out mice (FGFR3-/-) exhibited enhanced and prolonged endochondral bone growth accompanied by expansion of proliferating and hypertrophic chondrocytes within the cartilaginous growth plate. FGFR3 appeared to regulate endochondral ossification by an essentially negative mechanism .
Chen et al reported that FGF-FGFR3 signaling targets STATs and IHH to inhibit chondrocyte proliferation , while FGF-FGFR3 singnaling and PTHrP/IHH singaling may inhibit chondrocyte differentiation independently. It has been found that both Ach-(FGFR3 G380R) and TDII-type(FGFR3 K650E) mutant FGFR3 induce apoptotic changes and marked decrease of PTHrP expression in ATDC5 cells, a mouse chondrogenic cell line.PTHrP treatment can block the apoptotic response induced by FGFR3. Bone culture study has suggested that PTH1-34 inhibited differentiation and apoptosis of chondrocytes and improved bone growth,which indicates that PTH1-34 may be used as a potential therapeutic agent for achondroplasia.Whether or not the systemic administration of PTH1-34 is a pharmacological therapy for ACH needs to be further studied.
As a regulator of bone growth, FGFR3 is also involved in the regulation of fracture repair.Su et al reported that gain-of-function mutation of FGFR3 resulted in delayed fracture healing by inhibiting chondrocyte differentiation and bone resorption. Bone lengthening,a frequently performed surgery in patients with ACH, also involves fracture healing process. Thus ,searching for the biological measure to improve the bone healing in patients with ACH is very important.
Recombined human PTH1-34(Teriparatide) is the FDA-approved drug to treat osteoporosis in humans by promoting bone formation. There have been a variety of studies on the role of PTH1-34 in fracture healing,which demonstrated that PTH1-34 preferentially enhanced chondrocyte recruitment the rate of chondrocyte maturation to stimulate endochondral ossification. Whether or not PTH1-34 is an effective biological therapeutic measure for the delayed fracture repair in ACH needs further study.
In this study, we used mouse model mimicking human achondroplasia caused by gain-of-function mutation of FGFR3 (FGFR3G369C/+ mice) to explore the effect of PTH1-34 on the achondroplasia and delayed fracture healing resulting from ACH.
Methods
Part I Study of the effects of exogenous PTH1-34 on the bone development retardation of FGFR3G369C/+ mice
1. Animal model: New born wild-type(WT) and ACH mice were randomly divided into treatment group and control group. The mice in treatment group were given PTH1-34 80μg/(kg·d) subcutaneously until the end of the observation period while the control mice were given sterile water.
2. The body weight as well as length of naso-anal, tibia and femur were measured, whole skeleton staining and skull photography were performed. The cranial synchondrosis and foramen magnum of mice were observed.
3. Histological sections of mice at different ages were stained with HE and safranine-fast green . Chondrocyte proliferation in the growth plate was investigated by BrdU labeling assay.
4. The lengths of hypertrophic chondrocyte zone were measured. The expression of some marker genes during chondrocyte differentiation including typeⅡcollagen, type X collagen and PTHrP were detected by real-time PCR.
5. The bone culture system of metatarsal in vitro was established, metatarsal growth rates with or without PTH1-34 were determined and compared.
6. Intermittent PTH1-34 treatment were performed in primary cultured chondrocytes. Cell proliferation was detected using an in vitro MTT colorimetric assay and cytometry. The expression levels of FGFR3 were detected by real-time PCR and western blotting.
Part II Study of the effects of exogenous PTH1-34 on the delayed fracture healing of FGFR3G369C/+ mice
1. Closed fracture of proximal tibia was created and stabilized with an intramedullary pin in 7-8-week-old mice. The mice were randomly divided into treatment group and control group. The mice in treatment group were given PTH1-34 80μg/(kg·d) subcutaneously until the end of the observation period while the control mice were given sterile water.
2. Callus tissues were analyzed at 1-4 weeks post-fracture by radiography and histology.
3. The concentration of serum calcium and phosphorus were measured.
4. RNA was isolated from callus tissues, and the expression levels of bone formation-related genes were evaluated by real-time PCR.
Results
Part I Exogenous PTH can rescue the retarded bone development in ACH mice caused by carrying gain-of-function mutation of FGFR3
1. The naso-anal lengths of ACH mice treated by PTH1-34 at 4th week were significant longer than that in ACH control mice.The cranial synchondrosis time of ACH mice treated by PTH1-34 was delayed compared with that in ACH control mice.The size of foramen magnum had no significant difference.
2. The chondrocyte proliferative index measured by BrdU labeling assay indicated that the chondrocyte proliferative index in ACH mice treated by PTH1-34 was significantly higher than that in ACH control mice. The cell culture study confirmed this result.
3. On postnatal 7 d and 10 d, the hypertrophic zone of chondrocytes in growth plate of PTH1-34-treated ACH mice was wider than that in ACH control mice, and the secondary ossification center was observed advanced in PTH1-34 treated ACH mice..Real-time PCR measurement of both tissues and cells had comfirmed that.
4. Analysis suggested that the tibial growth rate of ACH mice was significantly slower than that in littermate WT control. PTH1-34-treatment can improve the growth suppression in ACH mice.
5. The expression of FGFR3 was decreased by PTH1-34 treatment.
Part II Exogenous PTH1-34 can rescue the delayed fracture healing caused by gain-of-function mutation of FGFR3 in mice
1. At 7 days post-fracture, cartilaginous callus areas were increased in PTH1-34-treated ACH mice compared with those of control mice. In contrast, at 14 days post-fracture, the remnant cartilaginous callus areas were smaller in PTH1-34-treated mice than those in control mice. There were remnant cartilaginous callus in ACH control mice while little remnant cartilaginous callus was observed at 21 days after fracture. The remnant fibrous bone areas in marrow cavity were also reduced in PTH1-34-treated mice compared to control mice at 28 days after fracture.
2. In the early stages of fracture (7 days), compared with the control group, the PCNA and COL10A1 mRNAs expression were obviously increased in PTH1-34 treatment group. In the middle stages of fracture(14 days), compared with the control group, the OC mRNA expression level were significantly increased in PTH1-34 treatment group
Conclusions:
1. The gross phenotypes of ACH mice treated with PTH1-34 including body weight and body length were increased compared with that in ACH control mice.
2. The proliferative and differentiation activity of chondrocytes in growth plates of ACH mice were inhanced by intermittent PTH1-34 treatment.
3. Results of PTH1-34 treatment on metatarsals suggested that PTH1-34 could promote the bone elongation of ACH mice.
4. The rescuing effect of PTH1-34 on bone retardation of ACH mice may be related to down regulation of FGFR3 expression.
5. Exogenous PTH1-34 promoted bone fracture healing in terms of increasing callus areas, endochondral ossification.
引文
1. Kronenberg, H.M., Developmental regulation of the growth plate. Nature, 2003. 423(6937): p. 332-6.
2. Ornitz, D.M., FGF signaling in the developing endochondral skeleton. Cytokine Growth Factor Rev, 2005. 16(2): p. 205-13.
3. Horton, W.A., J.G. Hall, and J.T. Hecht, Achondroplasia. Lancet, 2007. 370(9582): p. 162-72.
4. Dailey, L., et al., Mechanisms underlying differential responses to FGF signaling. Cytokine Growth Factor Rev, 2005. 16(2): p. 233-47.
5. Chen, L., et al., Gly369Cys mutation in mouse FGFR3 causes achondroplasia by affecting both chondrogenesis and osteogenesis. J Clin Invest, 1999. 104(11): p. 1517-25.
6. Chen, L., et al., A Ser(365)-->Cys mutation of fibroblast growth factor receptor 3 in mouse downregulates Ihh/PTHrP signals and causes severe achondroplasia. Hum Mol Genet, 2001. 10(5): p. 457-65.
7. Yamanaka, Y., et al., PTHrP rescues ATDC5 cells from apoptosis induced by FGF receptor 3 mutation. J Bone Miner Res, 2003. 18(8): p. 1395-403.
8. Ueda, K., et al., PTH has the potential to rescue disturbed bone growth in achondroplasia. Bone, 2007. 41(1): p. 13-8.
9. Nakajima, A., et al., Expression of fibroblast growth factor receptor-3 (FGFR3), signal transducer and activator of transcription-1, and cyclin-dependent kinase inhibitor p21 during endochondral ossification: differential role of FGFR3 in skeletal development and fracture repair. Endocrinology, 2003. 144(10): p. 4659-68.
10. Su, N., et al., Gain-of-function mutation of FGFR3 results in impaired fracture healing due to inhibition of chondrocyte differentiation. Biochem Biophys Res Commun, 2008. 376(3): p. 454-9.
11. Razzaque, M.S., et al., Conditional deletion of Indian hedgehog from collagen type 2alpha1-expressing cells results in abnormal endochondral bone formation. J Pathol, 2005. 207(4): p. 453-61.
12. Venkatesh, K.P., et al., Femoral lengthening in achondroplasia: magnitude of lengtheningin relation to patterns of callus, stiffness of adjacent joints and fracture. J Bone Joint Surg Br, 2009. 91(12): p. 1612-7.
13. Madore, G.R., P.J. Sherman, and J.M. Lane, Parathyroid hormone. J Am Acad Orthop Surg, 2004. 12(2): p. 67-71.
14. Andreassen, T.T., C. Ejersted, and H. Oxlund, Intermittent parathyroid hormone (1-34) treatment increases callus formation and mechanical strength of healing rat fractures. J Bone Miner Res, 1999. 14(6): p. 960-8.
15. Nakazawa, T., et al., Effects of low-dose, intermittent treatment with recombinant human parathyroid hormone (1-34) on chondrogenesis in a model of experimental fracture healing. Bone, 2005. 37(5): p. 711-9.
16. Ornitz, D.M. and P.J. Marie, FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease. Genes Dev, 2002. 16(12): p. 1446-65.
17. Deng, C., et al., Fibroblast growth factor receptor 3 is a negative regulator of bone growth. Cell, 1996. 84(6): p. 911-21.
18. Colvin, J.S., et al., Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3. Nat Genet, 1996. 12(4): p. 390-7.
19. Johnson, D.E. and L.T. Williams, Structural and functional diversity in the FGF receptor multigene family. Adv Cancer Res, 1993. 60: p. 1-41.
20. Karaplis, A.C. and D. Goltzman, PTH and PTHrP effects on the skeleton. Rev Endocr Metab Disord, 2000. 1(4): p. 331-41.
21. St-Jacques, B., M. Hammerschmidt, and A.P. McMahon, Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev, 1999. 13(16): p. 2072-86.
22. Naski, M.C., et al., Repression of hedgehog signaling and BMP4 expression in growth plate cartilage by fibroblast growth factor receptor 3. Development, 1998. 125(24): p. 4977-88.
23. Yang, C., et al., Effects of continuous and pulsatile PTH treatments on rat bone marrow stromal cells. Biochem Biophys Res Commun, 2009. 380(4): p. 791-6.
24. Schmidt, H., et al., C-type natriuretic peptide (CNP) is a bifurcation factor for sensory neurons. Proc Natl Acad Sci U S A, 2009. 106(39): p. 16847-52.
25. Matsushita, T., et al., FGFR3 promotes synchondrosis closure and fusion of ossification centers through the MAPK pathway. Hum Mol Genet, 2009. 18(2): p. 227-40.
26. Huang, M.S., et al., Hyperlipidemia impairs osteoanabolic effects of PTH. J Bone Miner Res, 2008. 23(10): p. 1672-9.
27. Su, N., et al., Gain-of-function mutation in FGFR3 in mice leads to decreased bone mass by affecting both osteoblastogenesis and osteoclastogenesis. Hum Mol Genet, 2010. 19(7): p. 1199-210.
28. Andreassen, T.T., et al., Increases in callus formation and mechanical strength of healing fractures in old rats treated with parathyroid hormone. Acta Orthop Scand, 2001. 72(3): p. 304-7.
29. Nakaoka, R., S.X. Hsiong, and D.J. Mooney, Regulation of chondrocyte differentiation level via co-culture with osteoblasts. Tissue Eng, 2006. 12(9): p. 2425-33.
30. Mauck, R.L., X. Yuan, and R.S. Tuan, Chondrogenic differentiation and functional maturation of bovine mesenchymal stem cells in long-term agarose culture. Osteoarthritis Cartilage, 2006. 14(2): p. 179-89.
31. Li, M., et al., FGFR3 down-regulates PTH/PTHrP receptor gene expression by mediating JAK/STAT signaling in chondrocytic cell line. J Electron Microsc (Tokyo), 2010.
32. Kronenberg, H.M., Developmental regulation of the growth plate. Nature, 2003. 423(6937): p. 332-336.
33. Colnot, C., Cellular and molecular interactions regulating skeletogenesis. J Cell Biochem, 2005. 95(4): p. 688-97.
34. Amizuka, N., H. Ozawa, and T. Sasaki, The biological action of parathyroid hormone-related peptide (PTHrP) and fibroblast growth factor receptor 3 (FGFR3) on bone and cartilage. Kaibogaku Zasshi, 2000. 75(5): p. 415-25.
35. Dailey, L., et al., Mechanisms underlying differential responses to FGF signaling. Cytokine Growth Factor Rev, 2005. 16(2): p. 233-247.
36. Keiper, G.L., Jr., B. Koch, and K.R. Crone, Achondroplasia and cervicomedullary compression: prospective evaluation and surgical treatment. Pediatr Neurosurg, 1999. 31(2): p. 78-83.
37. Liao, J.C., et al., Surgical treatment of achondroplasia with thoracolumbar kyphosis and spinal stenosis--a case report. Acta Orthop, 2006. 77(3): p. 541-4.
38. Shirley, E.D. and M.C. Ain, Achondroplasia: manifestations and treatment. J Am Acad Orthop Surg, 2009. 17(4): p. 231-41.
39. Ramaswami, U., et al., Treatment of achondroplasia with growth hormone: six years of experience. Pediatr Res, 1999. 46(4): p. 435-9.
40. Yamanaka, Y., et al., Molecular basis for the treatment of achondroplasia. Horm Res, 2003. 60 Suppl 3: p. 60-4.
41. Minina, E., et al., Interaction of FGF, Ihh/Pthlh, and BMP signaling integrates chondrocyte proliferation and hypertrophic differentiation. Dev Cell, 2002. 3(3): p. 439-49.
42. Enomoto-Iwamoto, M., et al., Hedgehog proteins stimulate chondrogenic cell differentiation and cartilage formation. J Bone Miner Res, 2000. 15(9): p. 1659-68.
43. Vortkamp, A., et al., Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science, 1996. 273(5275): p. 613-22.
44. Karsenty, G. and E.F. Wagner, Reaching a genetic and molecular understanding of skeletal development. Dev Cell, 2002. 2(4): p. 389-406.
45. Ogawa, T., et al., Human PTH (1-34) induces longitudinal bone growth in rats. J Bone Miner Metab, 2002. 20(2): p. 83-90.
46. Koike, T., et al., Potent mitogenic effects of parathyroid hormone (PTH) on embryonic chick and rabbit chondrocytes. Differential effects of age on growth, proteoglycan, and cyclic AMP responses of chondrocytes to PTH. J Clin Invest, 1990. 85(3): p. 626-31.
47. Crabb, I.D., et al., Differential effects of parathyroid hormone on chick growth plate and articular chondrocytes. Calcif Tissue Int, 1992. 50(1): p. 61-6.
48. Kobayashi, T., et al., PTHrP and Indian hedgehog control differentiation of growth plate chondrocytes at multiple steps. Development, 2002. 129(12): p. 2977-86.
49. Yang, Y., et al., Wnt5a and Wnt5b exhibit distinct activities in coordinating chondrocyte proliferation and differentiation. Development, 2003. 130(5): p. 1003-15.
50. Peters, K., et al., Unique expression pattern of the FGF receptor 3 gene during mouse organogenesis. Dev Biol, 1993. 155(2): p. 423-30.
51. Segev, O., et al., Restrained chondrocyte proliferation and maturation with abnormal growth plate vascularization and ossification in human FGFR-3(G380R) transgenic mice. Hum Mol Genet, 2000. 9(2): p. 249-58.
52. Amizuka, N., et al., Signalling by fibroblast growth factor receptor 3 and parathyroid hormone-related peptide coordinate cartilage and bone development. Bone, 2004. 34(1): p. 13-25.
53. Aarts, M.M., et al., Parathyroid hormone-related protein promotes quiescence and survival of serum-deprived chondrocytes by inhibiting rRNA synthesis. J Biol Chem, 2001. 276(41): p. 37934-43.
54. Amizuka, N., et al., Programmed cell death of chondrocytes and aberrant chondrogenesis in mice homozygous for parathyroid hormone-related peptide gene deletion. Endocrinology, 1996. 137(11): p. 5055-67.
55. Henderson, J.E., et al., Nucleolar localization of parathyroid hormone-related peptide enhances survival of chondrocytes under conditions that promote apoptotic cell death. Mol Cell Biol, 1995. 15(8): p. 4064-75.
56. Sahni, M., et al., STAT1 mediates the increased apoptosis and reduced chondrocyte proliferation in mice overexpressing FGF2. Development, 2001. 128(11): p. 2119-29.
57. Cheung, C., The future of bone healing. Clin Podiatr Med Surg, 2005. 22(4): p. 631-41 viii.
58. Branfoot, T., Research directions for bone healing. Injury, 2005. 36 Suppl 3: p. S51-4.
59. Le, A.X., et al., Molecular aspects of healing in stabilized and non-stabilized fractures. J Orthop Res, 2001. 19(1): p. 78-84.
60. Lanske, B., et al., PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science, 1996. 273(5275): p. 663-6.
61. Lieberman, J.R., A. Daluiski, and T.A. Einhorn, The role of growth factors in the repair of bone. Biology and clinical applications. J Bone Joint Surg Am, 2002. 84-A(6): p. 1032-44.
62. Shirley, E.D. and M.C. Ain, Achondroplasia: manifestations and treatment. J Am Acad Orthop Surg, 2009. 17(4): p. 231-41.
63. Spiro, A.S., et al., BMP-7-induced ectopic bone formation and fracture healing is impaired by systemic NSAID application in C57BL/6-mice. J Orthop Res, 2010. 28(6): p. 785-91.
64. Einhorn, T.A., Clinical applications of recombinant human BMPs: early experience and future development. J Bone Joint Surg Am, 2003. 85-A Suppl 3: p. 82-8.
65. Holzer, G., et al., Parathyroid hormone enhances fracture healing. A preliminary report. Clin Orthop Relat Res, 1999(366): p. 258-63
66. Thompson, Z., et al., A model for intramembranous ossification during fracture healing. J Orthop Res, 2002. 20(5): p. 1091-8.
67. Franceschi, R.T., Biological approaches to bone regeneration by gene therapy. J Dent Res, 2005. 84(12): p. 1093-103.
68. Neer, R.M., et al., Effect of parathyroid hormone (1-34) on fractures and bone mineral density in postmenopausal women with osteoporosis. N Engl J Med, 2001. 344(19): p. 1434-41.
69. Alkhiary, Y.M., et al., Enhancement of experimental fracture-healing by systemic administration of recombinant human parathyroid hormone (PTH 1-34). J Bone Joint Surg Am, 2005. 87(4): p. 731-41.
70. Andreassen, T.T., et al., Treatment with parathyroid hormone hPTH(1-34), hPTH(1-31), and monocyclic hPTH(1-31) enhances fracture strength and callus amount after withdrawal fracture strength and callus mechanical quality continue to increase. Calcif Tissue Int, 2004. 74(4): p. 351-6.
71. Barnes, G.L., et al., Stimulation of fracture-healing with systemic intermittent parathyroid hormone treatment. J Bone Joint Surg Am, 2008. 90 Suppl 1: p. 120-7.
1. Brewer, H.B., Jr. and R. Ronan, Bovine parathyroid hormone: amino acid sequence. Proc Natl Acad Sci U S A, 1970. 67(4): p. 1862-9.
2. Keutmann, H.T., et al., Complete amino acid sequence of human parathyroid hormone. Biochemistry, 1978. 17(26): p. 5723-9.
3.张克勤,甲状旁腺激素的分子生物学研究进展.中华医学杂志, 1993. 73(6): p. 369-372.
4. Neugebauer, W., et al., Structure and protein kinase C stimulating activities of lactam analogues of human parathyroid hormone fragment. Int J Pept Protein Res, 1994. 43(6): p. 555-62.
5. Barden, J.A. and B.E. Kemp, NMR solution structure of human parathyroid hormone(1-34). Biochemistry, 1993. 32(28): p. 7126-32.
6. Marx, U.C., et al., Solution structures of human parathyroid hormone fragments hPTH(1-34) and hPTH(1-39) and bovine parathyroid hormone fragment bPTH(1-37). Biochem Biophys Res Commun, 2000. 267(1): p. 213-20.
7. Gensure, R.C., T.J. Gardella, and H. Juppner, Multiple sites of contact between the carboxyl-terminal binding domain of PTHrP-(1--36) analogs and the amino-terminal extracellular domain of the PTH/PTHrP receptor identified by photoaffinity cross-linking. J Biol Chem, 2001. 276(31): p. 28650-8.
8. Hoare, S.R. and T.B. Usdin, Molecular mechanisms of ligand recognition by parathyroid hormone 1 (PTH1) and PTH2 receptors. Curr Pharm Des, 2001. 7(8): p. 689-713.
9. Hoare, S.R., et al., Evaluating the ligand specificity of zebrafish parathyroid hormone (PTH) receptors: comparison of PTH, PTH-related protein, and tuberoinfundibular peptide of 39 residues. Endocrinology, 2000. 141(9): p. 3080-6.
10. Divieti, P., et al., Receptors for the carboxyl-terminal region of pth(1-84) are highly expressed in osteocytic cells. Endocrinology, 2001. 142(2): p. 916-25.
11. Inomata, N., et al., Characterization of a novel parathyroid hormone (PTH) receptor with specificity for the carboxyl-terminal region of PTH-(1-84). Endocrinology, 1995. 136(11): p. 4732-40.
12. Dempster, D.W., et al., Effects of daily treatment with parathyroid hormone on bone microarchitecture and turnover in patients with osteoporosis: a paired biopsy study. J Bone Miner Res, 2001. 16(10): p. 1846-53.
13. Schluter, K.D., et al., The central part of parathyroid hormone stimulates thymidine incorporation of chondrocytes. J Biol Chem, 1989. 264(19): p. 11087-92.
14. Somjen, D., et al., Stimulation by defined parathyroid hormone fragments of cell proliferation in skeletal-derived cell cultures. Biochem J, 1990. 272(3): p. 781-5.
15. Dunlay, R. and K. Hruska, PTH receptor coupling to phospholipase C is an alternate pathway of signal transduction in bone and kidney. Am J Physiol, 1990. 258(2 Pt 2): p. F223-31.
16. Njeh CF, J.M., Genant HK Bone density and imaging of osteopororis. Endocrinology, 2005. 146(2): p. 1673–1695.
17. Delmas PD , C.R., Osteoporosis. Endocrinology, 2005. 146(2): p. 1751–1769.
18. Lindsay, R., et al., Randomised controlled study of effect of parathyroid hormone on vertebral-bone mass and fracture incidence among postmenopausal women on oestrogen with osteoporosis. Lancet, 1997. 350(9077): p. 550-5.
19. Roe EB, S.S., del Puerto GA, Parathyroid hormone 1–34 [hPTH(1–34)] and estrogen produce dramatic bone density increases in postmenopausal osteoporosis: results from a placebo-controlled randomized trial. J Bone Miner Res, 1999. 14(1): p. S137.
20. Neer, R.M., et al., Effect of parathyroid hormone (1-34) on fractures and bone mineral density in postmenopausal women with osteoporosis. N Engl J Med, 2001. 344(19): p. 1434-41.
21. Orwoll, E.S., et al., The effect of teriparatide [human parathyroid hormone (1-34)] therapy on bone density in men with osteoporosis. J Bone Miner Res, 2003. 18(1): p. 9-17.
22. Harada, S. and G.A. Rodan, Control of osteoblast function and regulation of bone mass. Nature, 2003. 423(6937): p. 349-55.
23. Mosekilde, L., et al., PTH has a more pronounced effect on vertebral bone mass and biomechanical competence than antiresorptive agents (estrogen and bisphosphonate) --assessed in sexually mature, ovariectomized rats. Bone, 1994. 15(4): p. 401-8.
24. Neer RM, Arnaud CD, Zanchetta JR, Prince R, Gaich GA, Reginster JY, HodsmanAB,Eriksen EF, Ish-Shalom S, Genant HK, Wang O, Mitlak BH. Effect of parathyroid hormone (1-34) on fractures and bone mineral density in postmenopausal womenwith osteoporosis. N Engl J Med. 2001;344:1434-41.
25. Lindsay R, Scheele WH, Neer R, Pohl G, Adami S, Mautalen C, Reginster JY, Stepan JJ, Myers SL, Mitlak BH. Sustained vertebral fracture risk reduction after withdrawal of teriparatide in postmenopausal women with osteoporosis. Arch InternMed. 2004; 164:2024-30.
26. Prince R, Sipos A, Hossain A, Syversen U, Ish-Shalom S, Marcinowska E, HalseJ, Lindsay R, Dalsky GP, Mitlak BH. Sustained nonvertebral fragility fracture riskreduction after discontinuation of teriparatide treatment. J Bone Miner Res.2005;20:1507-13.
27. Kaufman JM, Orwoll E, Goemaere S, San Martin J, Hossain A, Dalsky GP, Lindsay R, Mitlak BH. Teriparatide effects on vertebral fractures and bone mineraldensity in men with osteoporosis: treatment and discontinuation of therapy.Osteoporos Int. 2005;16:510-6.
28. Nakajima A, Shimoji N, Shiomi K, Shimizu S, Moriya H, Einhorn TA, YamazakiM. Mechanisms for the enhancement of fracture healing in rats treated with intermittent low-dose human parathyroid hormone (1-34). J Bone Miner Res.2002;17:2038-47.
29. Alkhiary YM, Gerstenfeld LC, Krall E, Westmore M, Sato M, Mitlak BH, EinhornTA. Enhancement of experimental fracture-healing by systemic administration of recombinant human parathyroid hormone (PTH 1-34). J Bone Joint Surg Am.2005;87:731-41.
30. Andreassen TT, Cacciafesta V. Intermittent parathyroid hormone treatment enhances guided bone regeneration in rat calvarial bone defects. J Craniofac Surg.2004;15:424-9.
31. Seebach C, Skripitz R, Andreassen TT, Aspenberg P. Intermittent parathyroid hormone (1-34) enhances mechanical strength and density of new bone after distraction osteogenesis in rats. J Orthop Res. 2004;22:472-8.
32. Hock JM, Krishnan V, Onyia JE, Bidwell JP, Milas J, Stanislaus D. Osteoblastapoptosis and bone turnover. J Bone Miner Res. 2001;16:975-84.
33. Hock JM. Anabolic actions of PTH in the skeletons of animals. J Musculoskelet Neuronal Interact. 2001;2:33-47.
34. Hodsman AB, Bauer DC, Dempster DW, Dian L, Hanley DA, Harris ST, Kendler DL, McClung MR, Miller PD, Olszynski WP, Orwell E, Yuen CK. Parathyroid hormone andteriparatide for the treatment of osteoporosis: a review of the evidence and suggested guidelines for its use. Endocr Rev. 2005;26:688-703.
35. Nakazawa T, Nakajima A, Shiomi K, Moriya H, Einhorn TA, Yamazaki M. Effects of low-dose, intermittent treatment with recombinant human parathyroid hormone (1-34) on chondrogenesis in a model of experimental fracture healing. Bone.2005;37:711-9.
36. Chung UI, Lanske B, Lee K, Li E, Kronenberg H. The parathyroid hormone/parathyroid hormone-related peptide receptor coordinates endochondral bone development by directly controlling chondrocyte differentiation. Proc Natl Acad SciU S A. 1998;95:13030-5.
37. Miao D, He B, Karaplis AC, Goltzman D. Parathyroid hormone is essential for normal fetal bone formation. J Clin Invest. 2002;109:1173-82.
38. Kakar S, Einhorn TA, Vora S, Miara LJ, Hon G, Wigner NA, Toben D, Jacobsen KA, Al-Sebaei MO, Song M, Trackman PC, Morgan EF, Gerstenfeld LC, Barnes GL.Enhanced chondrogenesis and Wnt-signaling in parathyroid hormone treated fractures. J Bone Miner Res. 2007;Aug 6.
39. Rodda SJ, McMahon AP. Distinct roles for Hedgehog and canonical Wnt signaling in specification, differentiation and maintenance of osteoblast progenitors. Development. 2006;133:3231-44.
2. Ornitz, D.M., FGF signaling in the developing endochondral skeleton. Cytokine Growth Factor Rev, 2005. 16(2): p. 205-13.
3. Horton, W.A., J.G. Hall, and J.T. Hecht, Achondroplasia. Lancet, 2007. 370(9582): p. 162-72.
4. Dailey, L., et al., Mechanisms underlying differential responses to FGF signaling. Cytokine Growth Factor Rev, 2005. 16(2): p. 233-47.
5. Chen, L., et al., Gly369Cys mutation in mouse FGFR3 causes achondroplasia by affecting both chondrogenesis and osteogenesis. J Clin Invest, 1999. 104(11): p. 1517-25.
6. Chen, L., et al., A Ser(365)-->Cys mutation of fibroblast growth factor receptor 3 in mouse downregulates Ihh/PTHrP signals and causes severe achondroplasia. Hum Mol Genet, 2001. 10(5): p. 457-65.
7. Yamanaka, Y., et al., PTHrP rescues ATDC5 cells from apoptosis induced by FGF receptor 3 mutation. J Bone Miner Res, 2003. 18(8): p. 1395-403.
8. Ueda, K., et al., PTH has the potential to rescue disturbed bone growth in achondroplasia. Bone, 2007. 41(1): p. 13-8.
9. Nakajima, A., et al., Expression of fibroblast growth factor receptor-3 (FGFR3), signal transducer and activator of transcription-1, and cyclin-dependent kinase inhibitor p21 during endochondral ossification: differential role of FGFR3 in skeletal development and fracture repair. Endocrinology, 2003. 144(10): p. 4659-68.
10. Su, N., et al., Gain-of-function mutation of FGFR3 results in impaired fracture healing due to inhibition of chondrocyte differentiation. Biochem Biophys Res Commun, 2008. 376(3): p. 454-9.
11. Razzaque, M.S., et al., Conditional deletion of Indian hedgehog from collagen type 2alpha1-expressing cells results in abnormal endochondral bone formation. J Pathol, 2005. 207(4): p. 453-61.
12. Venkatesh, K.P., et al., Femoral lengthening in achondroplasia: magnitude of lengtheningin relation to patterns of callus, stiffness of adjacent joints and fracture. J Bone Joint Surg Br, 2009. 91(12): p. 1612-7.
13. Madore, G.R., P.J. Sherman, and J.M. Lane, Parathyroid hormone. J Am Acad Orthop Surg, 2004. 12(2): p. 67-71.
14. Andreassen, T.T., C. Ejersted, and H. Oxlund, Intermittent parathyroid hormone (1-34) treatment increases callus formation and mechanical strength of healing rat fractures. J Bone Miner Res, 1999. 14(6): p. 960-8.
15. Nakazawa, T., et al., Effects of low-dose, intermittent treatment with recombinant human parathyroid hormone (1-34) on chondrogenesis in a model of experimental fracture healing. Bone, 2005. 37(5): p. 711-9.
16. Ornitz, D.M. and P.J. Marie, FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease. Genes Dev, 2002. 16(12): p. 1446-65.
17. Deng, C., et al., Fibroblast growth factor receptor 3 is a negative regulator of bone growth. Cell, 1996. 84(6): p. 911-21.
18. Colvin, J.S., et al., Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3. Nat Genet, 1996. 12(4): p. 390-7.
19. Johnson, D.E. and L.T. Williams, Structural and functional diversity in the FGF receptor multigene family. Adv Cancer Res, 1993. 60: p. 1-41.
20. Karaplis, A.C. and D. Goltzman, PTH and PTHrP effects on the skeleton. Rev Endocr Metab Disord, 2000. 1(4): p. 331-41.
21. St-Jacques, B., M. Hammerschmidt, and A.P. McMahon, Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev, 1999. 13(16): p. 2072-86.
22. Naski, M.C., et al., Repression of hedgehog signaling and BMP4 expression in growth plate cartilage by fibroblast growth factor receptor 3. Development, 1998. 125(24): p. 4977-88.
23. Yang, C., et al., Effects of continuous and pulsatile PTH treatments on rat bone marrow stromal cells. Biochem Biophys Res Commun, 2009. 380(4): p. 791-6.
24. Schmidt, H., et al., C-type natriuretic peptide (CNP) is a bifurcation factor for sensory neurons. Proc Natl Acad Sci U S A, 2009. 106(39): p. 16847-52.
25. Matsushita, T., et al., FGFR3 promotes synchondrosis closure and fusion of ossification centers through the MAPK pathway. Hum Mol Genet, 2009. 18(2): p. 227-40.
26. Huang, M.S., et al., Hyperlipidemia impairs osteoanabolic effects of PTH. J Bone Miner Res, 2008. 23(10): p. 1672-9.
27. Su, N., et al., Gain-of-function mutation in FGFR3 in mice leads to decreased bone mass by affecting both osteoblastogenesis and osteoclastogenesis. Hum Mol Genet, 2010. 19(7): p. 1199-210.
28. Andreassen, T.T., et al., Increases in callus formation and mechanical strength of healing fractures in old rats treated with parathyroid hormone. Acta Orthop Scand, 2001. 72(3): p. 304-7.
29. Nakaoka, R., S.X. Hsiong, and D.J. Mooney, Regulation of chondrocyte differentiation level via co-culture with osteoblasts. Tissue Eng, 2006. 12(9): p. 2425-33.
30. Mauck, R.L., X. Yuan, and R.S. Tuan, Chondrogenic differentiation and functional maturation of bovine mesenchymal stem cells in long-term agarose culture. Osteoarthritis Cartilage, 2006. 14(2): p. 179-89.
31. Li, M., et al., FGFR3 down-regulates PTH/PTHrP receptor gene expression by mediating JAK/STAT signaling in chondrocytic cell line. J Electron Microsc (Tokyo), 2010.
32. Kronenberg, H.M., Developmental regulation of the growth plate. Nature, 2003. 423(6937): p. 332-336.
33. Colnot, C., Cellular and molecular interactions regulating skeletogenesis. J Cell Biochem, 2005. 95(4): p. 688-97.
34. Amizuka, N., H. Ozawa, and T. Sasaki, The biological action of parathyroid hormone-related peptide (PTHrP) and fibroblast growth factor receptor 3 (FGFR3) on bone and cartilage. Kaibogaku Zasshi, 2000. 75(5): p. 415-25.
35. Dailey, L., et al., Mechanisms underlying differential responses to FGF signaling. Cytokine Growth Factor Rev, 2005. 16(2): p. 233-247.
36. Keiper, G.L., Jr., B. Koch, and K.R. Crone, Achondroplasia and cervicomedullary compression: prospective evaluation and surgical treatment. Pediatr Neurosurg, 1999. 31(2): p. 78-83.
37. Liao, J.C., et al., Surgical treatment of achondroplasia with thoracolumbar kyphosis and spinal stenosis--a case report. Acta Orthop, 2006. 77(3): p. 541-4.
38. Shirley, E.D. and M.C. Ain, Achondroplasia: manifestations and treatment. J Am Acad Orthop Surg, 2009. 17(4): p. 231-41.
39. Ramaswami, U., et al., Treatment of achondroplasia with growth hormone: six years of experience. Pediatr Res, 1999. 46(4): p. 435-9.
40. Yamanaka, Y., et al., Molecular basis for the treatment of achondroplasia. Horm Res, 2003. 60 Suppl 3: p. 60-4.
41. Minina, E., et al., Interaction of FGF, Ihh/Pthlh, and BMP signaling integrates chondrocyte proliferation and hypertrophic differentiation. Dev Cell, 2002. 3(3): p. 439-49.
42. Enomoto-Iwamoto, M., et al., Hedgehog proteins stimulate chondrogenic cell differentiation and cartilage formation. J Bone Miner Res, 2000. 15(9): p. 1659-68.
43. Vortkamp, A., et al., Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science, 1996. 273(5275): p. 613-22.
44. Karsenty, G. and E.F. Wagner, Reaching a genetic and molecular understanding of skeletal development. Dev Cell, 2002. 2(4): p. 389-406.
45. Ogawa, T., et al., Human PTH (1-34) induces longitudinal bone growth in rats. J Bone Miner Metab, 2002. 20(2): p. 83-90.
46. Koike, T., et al., Potent mitogenic effects of parathyroid hormone (PTH) on embryonic chick and rabbit chondrocytes. Differential effects of age on growth, proteoglycan, and cyclic AMP responses of chondrocytes to PTH. J Clin Invest, 1990. 85(3): p. 626-31.
47. Crabb, I.D., et al., Differential effects of parathyroid hormone on chick growth plate and articular chondrocytes. Calcif Tissue Int, 1992. 50(1): p. 61-6.
48. Kobayashi, T., et al., PTHrP and Indian hedgehog control differentiation of growth plate chondrocytes at multiple steps. Development, 2002. 129(12): p. 2977-86.
49. Yang, Y., et al., Wnt5a and Wnt5b exhibit distinct activities in coordinating chondrocyte proliferation and differentiation. Development, 2003. 130(5): p. 1003-15.
50. Peters, K., et al., Unique expression pattern of the FGF receptor 3 gene during mouse organogenesis. Dev Biol, 1993. 155(2): p. 423-30.
51. Segev, O., et al., Restrained chondrocyte proliferation and maturation with abnormal growth plate vascularization and ossification in human FGFR-3(G380R) transgenic mice. Hum Mol Genet, 2000. 9(2): p. 249-58.
52. Amizuka, N., et al., Signalling by fibroblast growth factor receptor 3 and parathyroid hormone-related peptide coordinate cartilage and bone development. Bone, 2004. 34(1): p. 13-25.
53. Aarts, M.M., et al., Parathyroid hormone-related protein promotes quiescence and survival of serum-deprived chondrocytes by inhibiting rRNA synthesis. J Biol Chem, 2001. 276(41): p. 37934-43.
54. Amizuka, N., et al., Programmed cell death of chondrocytes and aberrant chondrogenesis in mice homozygous for parathyroid hormone-related peptide gene deletion. Endocrinology, 1996. 137(11): p. 5055-67.
55. Henderson, J.E., et al., Nucleolar localization of parathyroid hormone-related peptide enhances survival of chondrocytes under conditions that promote apoptotic cell death. Mol Cell Biol, 1995. 15(8): p. 4064-75.
56. Sahni, M., et al., STAT1 mediates the increased apoptosis and reduced chondrocyte proliferation in mice overexpressing FGF2. Development, 2001. 128(11): p. 2119-29.
57. Cheung, C., The future of bone healing. Clin Podiatr Med Surg, 2005. 22(4): p. 631-41 viii.
58. Branfoot, T., Research directions for bone healing. Injury, 2005. 36 Suppl 3: p. S51-4.
59. Le, A.X., et al., Molecular aspects of healing in stabilized and non-stabilized fractures. J Orthop Res, 2001. 19(1): p. 78-84.
60. Lanske, B., et al., PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science, 1996. 273(5275): p. 663-6.
61. Lieberman, J.R., A. Daluiski, and T.A. Einhorn, The role of growth factors in the repair of bone. Biology and clinical applications. J Bone Joint Surg Am, 2002. 84-A(6): p. 1032-44.
62. Shirley, E.D. and M.C. Ain, Achondroplasia: manifestations and treatment. J Am Acad Orthop Surg, 2009. 17(4): p. 231-41.
63. Spiro, A.S., et al., BMP-7-induced ectopic bone formation and fracture healing is impaired by systemic NSAID application in C57BL/6-mice. J Orthop Res, 2010. 28(6): p. 785-91.
64. Einhorn, T.A., Clinical applications of recombinant human BMPs: early experience and future development. J Bone Joint Surg Am, 2003. 85-A Suppl 3: p. 82-8.
65. Holzer, G., et al., Parathyroid hormone enhances fracture healing. A preliminary report. Clin Orthop Relat Res, 1999(366): p. 258-63
66. Thompson, Z., et al., A model for intramembranous ossification during fracture healing. J Orthop Res, 2002. 20(5): p. 1091-8.
67. Franceschi, R.T., Biological approaches to bone regeneration by gene therapy. J Dent Res, 2005. 84(12): p. 1093-103.
68. Neer, R.M., et al., Effect of parathyroid hormone (1-34) on fractures and bone mineral density in postmenopausal women with osteoporosis. N Engl J Med, 2001. 344(19): p. 1434-41.
69. Alkhiary, Y.M., et al., Enhancement of experimental fracture-healing by systemic administration of recombinant human parathyroid hormone (PTH 1-34). J Bone Joint Surg Am, 2005. 87(4): p. 731-41.
70. Andreassen, T.T., et al., Treatment with parathyroid hormone hPTH(1-34), hPTH(1-31), and monocyclic hPTH(1-31) enhances fracture strength and callus amount after withdrawal fracture strength and callus mechanical quality continue to increase. Calcif Tissue Int, 2004. 74(4): p. 351-6.
71. Barnes, G.L., et al., Stimulation of fracture-healing with systemic intermittent parathyroid hormone treatment. J Bone Joint Surg Am, 2008. 90 Suppl 1: p. 120-7.
1. Brewer, H.B., Jr. and R. Ronan, Bovine parathyroid hormone: amino acid sequence. Proc Natl Acad Sci U S A, 1970. 67(4): p. 1862-9.
2. Keutmann, H.T., et al., Complete amino acid sequence of human parathyroid hormone. Biochemistry, 1978. 17(26): p. 5723-9.
3.张克勤,甲状旁腺激素的分子生物学研究进展.中华医学杂志, 1993. 73(6): p. 369-372.
4. Neugebauer, W., et al., Structure and protein kinase C stimulating activities of lactam analogues of human parathyroid hormone fragment. Int J Pept Protein Res, 1994. 43(6): p. 555-62.
5. Barden, J.A. and B.E. Kemp, NMR solution structure of human parathyroid hormone(1-34). Biochemistry, 1993. 32(28): p. 7126-32.
6. Marx, U.C., et al., Solution structures of human parathyroid hormone fragments hPTH(1-34) and hPTH(1-39) and bovine parathyroid hormone fragment bPTH(1-37). Biochem Biophys Res Commun, 2000. 267(1): p. 213-20.
7. Gensure, R.C., T.J. Gardella, and H. Juppner, Multiple sites of contact between the carboxyl-terminal binding domain of PTHrP-(1--36) analogs and the amino-terminal extracellular domain of the PTH/PTHrP receptor identified by photoaffinity cross-linking. J Biol Chem, 2001. 276(31): p. 28650-8.
8. Hoare, S.R. and T.B. Usdin, Molecular mechanisms of ligand recognition by parathyroid hormone 1 (PTH1) and PTH2 receptors. Curr Pharm Des, 2001. 7(8): p. 689-713.
9. Hoare, S.R., et al., Evaluating the ligand specificity of zebrafish parathyroid hormone (PTH) receptors: comparison of PTH, PTH-related protein, and tuberoinfundibular peptide of 39 residues. Endocrinology, 2000. 141(9): p. 3080-6.
10. Divieti, P., et al., Receptors for the carboxyl-terminal region of pth(1-84) are highly expressed in osteocytic cells. Endocrinology, 2001. 142(2): p. 916-25.
11. Inomata, N., et al., Characterization of a novel parathyroid hormone (PTH) receptor with specificity for the carboxyl-terminal region of PTH-(1-84). Endocrinology, 1995. 136(11): p. 4732-40.
12. Dempster, D.W., et al., Effects of daily treatment with parathyroid hormone on bone microarchitecture and turnover in patients with osteoporosis: a paired biopsy study. J Bone Miner Res, 2001. 16(10): p. 1846-53.
13. Schluter, K.D., et al., The central part of parathyroid hormone stimulates thymidine incorporation of chondrocytes. J Biol Chem, 1989. 264(19): p. 11087-92.
14. Somjen, D., et al., Stimulation by defined parathyroid hormone fragments of cell proliferation in skeletal-derived cell cultures. Biochem J, 1990. 272(3): p. 781-5.
15. Dunlay, R. and K. Hruska, PTH receptor coupling to phospholipase C is an alternate pathway of signal transduction in bone and kidney. Am J Physiol, 1990. 258(2 Pt 2): p. F223-31.
16. Njeh CF, J.M., Genant HK Bone density and imaging of osteopororis. Endocrinology, 2005. 146(2): p. 1673–1695.
17. Delmas PD , C.R., Osteoporosis. Endocrinology, 2005. 146(2): p. 1751–1769.
18. Lindsay, R., et al., Randomised controlled study of effect of parathyroid hormone on vertebral-bone mass and fracture incidence among postmenopausal women on oestrogen with osteoporosis. Lancet, 1997. 350(9077): p. 550-5.
19. Roe EB, S.S., del Puerto GA, Parathyroid hormone 1–34 [hPTH(1–34)] and estrogen produce dramatic bone density increases in postmenopausal osteoporosis: results from a placebo-controlled randomized trial. J Bone Miner Res, 1999. 14(1): p. S137.
20. Neer, R.M., et al., Effect of parathyroid hormone (1-34) on fractures and bone mineral density in postmenopausal women with osteoporosis. N Engl J Med, 2001. 344(19): p. 1434-41.
21. Orwoll, E.S., et al., The effect of teriparatide [human parathyroid hormone (1-34)] therapy on bone density in men with osteoporosis. J Bone Miner Res, 2003. 18(1): p. 9-17.
22. Harada, S. and G.A. Rodan, Control of osteoblast function and regulation of bone mass. Nature, 2003. 423(6937): p. 349-55.
23. Mosekilde, L., et al., PTH has a more pronounced effect on vertebral bone mass and biomechanical competence than antiresorptive agents (estrogen and bisphosphonate) --assessed in sexually mature, ovariectomized rats. Bone, 1994. 15(4): p. 401-8.
24. Neer RM, Arnaud CD, Zanchetta JR, Prince R, Gaich GA, Reginster JY, HodsmanAB,Eriksen EF, Ish-Shalom S, Genant HK, Wang O, Mitlak BH. Effect of parathyroid hormone (1-34) on fractures and bone mineral density in postmenopausal womenwith osteoporosis. N Engl J Med. 2001;344:1434-41.
25. Lindsay R, Scheele WH, Neer R, Pohl G, Adami S, Mautalen C, Reginster JY, Stepan JJ, Myers SL, Mitlak BH. Sustained vertebral fracture risk reduction after withdrawal of teriparatide in postmenopausal women with osteoporosis. Arch InternMed. 2004; 164:2024-30.
26. Prince R, Sipos A, Hossain A, Syversen U, Ish-Shalom S, Marcinowska E, HalseJ, Lindsay R, Dalsky GP, Mitlak BH. Sustained nonvertebral fragility fracture riskreduction after discontinuation of teriparatide treatment. J Bone Miner Res.2005;20:1507-13.
27. Kaufman JM, Orwoll E, Goemaere S, San Martin J, Hossain A, Dalsky GP, Lindsay R, Mitlak BH. Teriparatide effects on vertebral fractures and bone mineraldensity in men with osteoporosis: treatment and discontinuation of therapy.Osteoporos Int. 2005;16:510-6.
28. Nakajima A, Shimoji N, Shiomi K, Shimizu S, Moriya H, Einhorn TA, YamazakiM. Mechanisms for the enhancement of fracture healing in rats treated with intermittent low-dose human parathyroid hormone (1-34). J Bone Miner Res.2002;17:2038-47.
29. Alkhiary YM, Gerstenfeld LC, Krall E, Westmore M, Sato M, Mitlak BH, EinhornTA. Enhancement of experimental fracture-healing by systemic administration of recombinant human parathyroid hormone (PTH 1-34). J Bone Joint Surg Am.2005;87:731-41.
30. Andreassen TT, Cacciafesta V. Intermittent parathyroid hormone treatment enhances guided bone regeneration in rat calvarial bone defects. J Craniofac Surg.2004;15:424-9.
31. Seebach C, Skripitz R, Andreassen TT, Aspenberg P. Intermittent parathyroid hormone (1-34) enhances mechanical strength and density of new bone after distraction osteogenesis in rats. J Orthop Res. 2004;22:472-8.
32. Hock JM, Krishnan V, Onyia JE, Bidwell JP, Milas J, Stanislaus D. Osteoblastapoptosis and bone turnover. J Bone Miner Res. 2001;16:975-84.
33. Hock JM. Anabolic actions of PTH in the skeletons of animals. J Musculoskelet Neuronal Interact. 2001;2:33-47.
34. Hodsman AB, Bauer DC, Dempster DW, Dian L, Hanley DA, Harris ST, Kendler DL, McClung MR, Miller PD, Olszynski WP, Orwell E, Yuen CK. Parathyroid hormone andteriparatide for the treatment of osteoporosis: a review of the evidence and suggested guidelines for its use. Endocr Rev. 2005;26:688-703.
35. Nakazawa T, Nakajima A, Shiomi K, Moriya H, Einhorn TA, Yamazaki M. Effects of low-dose, intermittent treatment with recombinant human parathyroid hormone (1-34) on chondrogenesis in a model of experimental fracture healing. Bone.2005;37:711-9.
36. Chung UI, Lanske B, Lee K, Li E, Kronenberg H. The parathyroid hormone/parathyroid hormone-related peptide receptor coordinates endochondral bone development by directly controlling chondrocyte differentiation. Proc Natl Acad SciU S A. 1998;95:13030-5.
37. Miao D, He B, Karaplis AC, Goltzman D. Parathyroid hormone is essential for normal fetal bone formation. J Clin Invest. 2002;109:1173-82.
38. Kakar S, Einhorn TA, Vora S, Miara LJ, Hon G, Wigner NA, Toben D, Jacobsen KA, Al-Sebaei MO, Song M, Trackman PC, Morgan EF, Gerstenfeld LC, Barnes GL.Enhanced chondrogenesis and Wnt-signaling in parathyroid hormone treated fractures. J Bone Miner Res. 2007;Aug 6.
39. Rodda SJ, McMahon AP. Distinct roles for Hedgehog and canonical Wnt signaling in specification, differentiation and maintenance of osteoblast progenitors. Development. 2006;133:3231-44.