Polycytosine RNA-binding protein 1 regulates osteoblast function via a ferroptosis pathway in type 2 diabetic osteoporosis

BACKGROUND Recently,type 2 diabetic osteoporosis(T2DOP)has become a research hotspot for the complications of diabetes,but the specific mechanism of its occurrence and development remains unknown.Ferroptosis caused by iron overload is con-sidered an important cause of T2DOP.Polycytosine RNA-binding protein 1(PCBP1),an iron ion chaperone,is considered a protector of ferroptosis.AIM To investigate the existence of ferroptosis and specific role of PCBP1 in the development of type 2 diabetes.METHODS A cell counting kit-8 assay was used to detect changes in osteoblast viability under high glucose(HG)and/or ferroptosis inhibitors at different concentrations and times.Transmission electron microscopy was used to examine the morpho-logical changes in the mitochondria of osteoblasts under HG,and western blotting was used to detect the expression levels of PCBP1,ferritin,and the ferroptosis-related protein glutathione peroxidase 4(GPX4).A lentivirus silenced and overex-pressed PCBP1.Western blotting was used to detect the expression levels of the osteoblast functional proteins osteoprotegerin(OPG)and osteocalcin(OCN),whereas flow cytometry was used to detect changes in reactive oxygen species(ROS)levels in each group.RESULTS Under HG,the viability of osteoblasts was considerably decreased,the number of mitochondria undergoing atrophy was considerably increased,PCBP1 and ferritin expression levels were increased,and GPX4 expression was decreased.Western blotting results demonstrated that infection with lentivirus overexpressing PCBP1,increased the expression levels of ferritin,GPX4,OPG,and OCN,compared with the HG group.Flow cytometry results showed a reduction in ROS,and an opposite result was obtained after silencing PCBP1.CONCLUSION PCBP1 may protect osteoblasts and reduce the harm caused by ferroptosis by promoting ferritin expression under a HG environment.Moreover,PCBP1 may be a potential therapeutic target for T2DOP.

Nystose attenuates bone loss and promotes BMSCs differentiation to osteoblasts through BMP and Wnt/β-catenin pathway in ovariectomized mice

Increasing the osteogenic differentiation ability and decreasing the adipogenic differentiation ability of bone marrow mesenchymal stem cells(BMSCs)is a potential strategy for the treatment of osteoporosis(OP).Naturally derived oligosaccharides have shown significant anti-osteoporotic effects.Nystose(NST),an oligosaccharide,was isolated from the roots of Morinda officinalis How.(MO).The aim of the present study was to investigate the effects of NST on bone loss in ovariectomized mice,and explore the underlying mechanism of NST in promoting differentiation of BMSCs to osteoblasts.Administration of NST(40,80 and 160 mg/kg)and the positive control of estradiol valerate(0.2 mg/kg)for 8 weeks significantly prevented bone loss induced by ovariectomy(OVX),increased the bone mass density(BMD),improved the bone microarchitecture and reduced urine calcium and deoxypyridinoline(DPD)in ovariectomized mice,while inhibited the increase of body weight without significantly affecting the uterus weight.Furthermore,we found that NST increased osteogenic differentiation,inhibited adipogenic differentiation of BMSCs in vitro,and upregulated the expression of the key proteins of BMP and Wnt/β-catenin pathways.In addition,Noggin and Dickkopf-related protein-1(DKK-1)reversed the effect of NST on osteogenic differentiation and expression of the key proteins in BMP and Wnt/β-catenin pathway.The luciferase activities and the molecular docking analysis further supported the mechanism of NST.In conclusion,these results indicating that NST can be clinically used as a potential alternative medicine for the prevention and treatment of postmenopausal osteoporosis.

Establishment and evaluation of the primary cultured tibial osteoblast model of broiler chicks

Osteoblasts are considered as a major factor contributing to bone development and mineralization,however,few studies have been done to establish and evaluate the primary cultured tibial osteoblast model of broiler chicks.Therefore,in the present study,two experiments were conducted to establish and evaluate the primary cultured tibial osteoblast model of broiler chicks.In experiment 1,osteoblasts were isolated from the tibia of one-day-old Arbor Acre male broiler chicks using the explant method and identified through the cell morphology,alkaline phosphatase(ALP)and alizarin red staining.Experiment 2 was carried out to evaluate the vitality and mineralization of primary cultured tibial osteoblasts of broilers on days 4,8,12,16,20,24,28 and 32 after incubation,respectively.The results from experiment 1 demonstrated that primary cultured tibial osteoblasts of broilers showed a spindle-shaped,triangular or polygonal morphology.More than 95%of the cells were stained blue-black after ALP staining,and mineralized nodules were formed after 4 days of continuous incubation.In experiment 2,lactate dehydrogenase(LDH)activity stayed at a relatively stabilized level although incubation time affected(P=0.0012)it during the whole culture period.Additionally,incubation time affected(P≤0.0001)the number and proportion of the area of mineralized nodules.They increased linearly and quadratically(P<0.04)with the increase of incubation time,and remained at a stabilized level from 24 to 32 days of incubation.The estimates of the optimal incubation time were 17 and 26 days based on the best fitted broken-line or quadratic models(P<0.0001)of the number and proportion of the area of mineralized nodules,respectively.These results indicate that the primary cultured tibial osteoblast model of broilers has been established successfully by the explant method,and it showed typical osteoblast morphology and characteristics of ALP activity and mineralization,and could maintain a relatively stabilized vitality from 4 to 32 days of incubation;and the optimal incubation time of primary tibial osteoblasts was 17 to 26 days.Therefore,it could be used to further study the underlying mechanisms of bone development and mineralization of broiler chicks.

Effects of areca nut consumption on cell differentiation of osteoblasts, myoblasts, and fibroblasts

Areca nut is used worldwide as a hallucinogenic addicting drug along the tropical belt.Arecoline,a toxic compound,is the most important alkaloid in areca nuts.The adverse effects of oral uptake and chewing of areca nut are well known.For example,the possibility of cancer caused by chewing areca nuts is widely discussed.Chewing areca nut has other adverse effects on other organs,including abnormal cell differentiation,oral cancer,and several other diseases.The use of areca nut is also associated with low birthweight.Skeletal musculature is the largest organ in the body and is attached to the bones.During embryo development,the differentiation of bone and muscle cells is critical.In this article,we reviewed the effects of areca nut and arecoline on embryonic cell differentiation,particularly osteoblasts,myoblasts,and fibroblasts.

Lipolysis supports bone formation by providing osteoblasts with endogenous fatty acid substrates to maintain bioenergetic status

Bone formation is a highly energy-demanding process that can be impacted by metabolic disorders.Glucose has been considered the principal substrate for osteoblasts,although fatty acids are also important for osteoblast function.Here,we report that osteoblasts can derive energy from endogenous fatty acids stored in lipid droplets via lipolysis and that this process is critical for bone formation.As such,we demonstrate that osteoblasts accumulate lipid droplets that are highly dynamic and provide the molecular mechanism by which they serve as a fuel source for energy generation during osteoblast maturation.Inhibiting cytoplasmic lipolysis leads to both an increase in lipid droplet size in osteoblasts and an impairment in osteoblast function.The fatty acids released by lipolysis from these lipid droplets become critical for cellular energy production as cellular energetics shifts towards oxidative phosphorylation during nutrient-depleted conditions.In vivo,conditional deletion of the ATGL-encoding gene Pnpla2 in osteoblast progenitor cells reduces cortical and trabecular bone parameters and alters skeletal lipid metabolism.Collectively,our data demonstrate that osteoblasts store fatty acids in the form of lipid droplets,which are released via lipolysis to support cellular bioenergetic status when nutrients are limited.Perturbations in this process result in impairment of bone formation,specifically reducing ATP production and overall osteoblast function.

Effects of acupotomy on the activity of osteoclasts and osteoblasts in the subchondral bone of rabbits with early and mid-stage knee osteoarthritis models

Objective:To investigate whether acupotomy could inhibit subchondral bone remodeling in knee osteoarthritis(KOA)rabbits by regulating the activity of osteoblasts and osteoclasts.Methods:KOA rabbits were prepared by immobilization for 6 and 9 weeks by Videman method.Nine groups of rabbits(control,6 weeks and 9 weeks model,6 weeks and 9 weeks acupotomy,6 weeks and 9 weeks electroacupuncture,and 6 weeks and 9 weeks drug groups)received acupotomy,electroacupuncture and risedronate sodium intervention,respectively,for 3 weeks.Results:Acupotomy can inhibit the activity of osteoclasts and osteoblasts in subchondral bone by reducing the proteins expression of cathepsin K(CK)and tartrate-resistant acid phosphatase(TRAP)and decreasing the proteins expression of osteocalcin(OCN)and alkaline phosphatase(ALP),to intercept the abnormal bone resorption and bone formation of subchondral bone in 6-week and 9-week immobilization-induced KOA rabbits.Conclusion:These findings indicated that acupotomy may be more advantageous than risedronate sodium intervention in modulating subchondral bone remodeling in KOA rabbits,especially in 9-week immobilization-induced KOA rabbits.

The art of building bone: emerging role of chondrocyte-to-osteoblast transdifferentiation in endochondral ossification

There is a worldwide epidemic of skeletal diseases causing not only a public health issue but also accounting for a sizable portion of healthcare expenditures. The vertebrate skeleton is known to be formed by mesenchymal cells condensing into tissue elements(patterning phase) followed by their differentiation into cartilage(chondrocytes) or bone(osteoblasts) cells within the condensations. During the growth and remodeling phase, bone is formed directly via intramembranous ossification or through a cartilage to bone conversion via endochondral ossification routes. The canonical pathway of the endochondral bone formation process involves apoptosis of hypertrophic chondrocytes followed by vascular invasion that brings in osteoclast precursors to remove cartilage and osteoblast precursors to form bone. However, there is now an emerging role for chondrocyte-to-osteoblast transdifferentiation in the endochondral ossification process. Although the concept of "transdifferentiation" per se is not recent,new data using a variety of techniques to follow the fate of chondrocytes in different bones during embryonic and post-natal growth as well as during fracture repair in adults have identified three different models for chondrocyte-to-osteoblast transdifferentiation(direct transdifferentiation, dedifferentiation to redifferentiation, and chondrocyte to osteogenic precursor). This review focuses on the emerging models of chondrocyte-to-osteoblast transdifferentiation and their implications for the treatment of skeletal diseases as well as the possible signaling pathways that contribute to chondrocyte-to-osteoblast transdifferentiation processes.

Paracrine and endocrine actions of bone——the functions of secretory proteins from osteoblasts, osteocytes, and osteoclasts

The skeleton is a dynamic organ that is constantly remodeled. Proteins secreted from bone cells, namely osteoblasts, osteocytes,and osteoclasts exert regulation on osteoblastogenesis, osteclastogenesis, and angiogenesis in a paracrine manner. Osteoblasts secrete a range of different molecules including RANKL/OPG, M-CSF, SEMA3A, WNT5A, and WNT16 that regulate osteoclastogenesis. Osteoblasts also produce VEGFA that stimulates osteoblastogenesis and angiogenesis. Osteocytes produce sclerostin（SOST） that inhibits osteoblast differentiation and promotes osteoclast differentiation. Osteoclasts secrete factors including BMP6, CTHRC1, EFNB2, S1P, WNT10B, SEMA4D, and CT-1 that act on osteoblasts and osteocytes, and thereby influencea A osteogenesis. Osteoclast precursors produce the angiogenic factor PDGF-BB to promote the formation of Type H vessels, which then stimulate osteoblastogenesis. Besides, the evidences over the past decades show that at least three hormones or ＂osteokines＂from bone cells have endocrine functions. FGF23 is produced by osteoblasts and osteocytes and can regulate phosphate metabolism. Osteocalcin（OCN） secreted by osteoblasts regulates systemic glucose and energy metabolism, reproduction, and cognition. Lipocalin-2（LCN2） is secreted by osteoblasts and can influence energy metabolism by suppressing appetite in the brain.We review the recent progresses in the paracrine and endocrine functions of the secretory proteins of osteoblasts, osteocytes, and osteoclasts, revealing connections of the skeleton with other tissues and providing added insights into the pathogenesis of degenerative diseases affecting multiple organs and the drug discovery process.

Modulation of Isoflavones on Bone-nodule Formation in Rat Calvaria Osteoblasts in vitro

Objective To observe the effects of two main isoflavones, daidzein and genistein on the bone-nodule formation in rat calvaria osteoblasts in vitro. Methods Osteoblasts obtained from newborn Sprague-dawley rat calvarias were cultured for several generations. The second generation cells were cultured in Minimum Essential Medium supplemented with ascorbic acid and Na-beta-glycerophosphate for several days, in the presence of daidzein and genistein, with or without the estrogen receptor antagonist ICI 182780. Number of nodules was counted at the end of the incubation period (day 20) by staining with Alizarin Red S calcium stain. The release of osteocalcin, as a marker of osteoblast activity, was also determined on day 7 and day 12 during the incubation period. Results Compared with the control, the numbers of nodules were both increased by incubation with daidzein and genistein. 17β-estradiol was used as a positive control and proved to be a more effective inducer of the increase in bone-nodules formation than daidzein and genistein. The release of osteocalcin into culture media was also increased in the presence of daidzein and genistein, as well as 17β-estradiol on day 7 and day 12 (day 12 were higher). The estrogen receptor antagonist ICI 182780 completely blocked the genistein- and 17β-estradiol-induced increase of nodule numbers and osteocalcin release in osteoblasts. However, the effects induced by daidzein could not be inhibited by ICI 182780. Conclusion These findings suggest that geinistein can stimulate bone-nodule formation and increase the release of osteocalcin in rat osteoblasts. The effects, like those induced by 17β-estradiol, are mediated by the estrogen receptor dependent pathway. Daidzein also can stimulate bone-nodule formation and increase the release of osteocalcin in rat osteoblasts, but it is not, at least not merely, mediated by the estrogen receptor dependent pathway.

TGF-β and BMP signaling in osteoblast,skeletal development,and bone formation,homeostasis and disease

INTRODUCTIONThe transforming growth factor-β （TGF-β） superfamily com- prises TGF-βs, Activin, bone morphogenetic proteins （BMPs） and other related proteins. TGF-β superfamily members act through a heteromeric receptor complex,, comprised of type I and type II receptors at the cell surface that transduce intracellular signals via Smad complex or mitogen-activated protein kinase （MAPK） cascade.

New roles of osteoblasts involved in osteoclast differentiation

Bone-resorbing osteoclasts are formed from a monocyte/macrophage lineage under the strict control o bone-forming osteoblasts. So far,macrophage colonystimulating factor(M-CSF),receptor activator o nuclear factor-κB ligand(RANKL),and osteoprotegerin(OPG) produced by osteoblasts play major roles in the regulation of osteoclast differentiation. Recent studies have shown that osteoblasts regulate osteoclastogenesis through several mechanisms independent o M-CSF,RANKL,and OPG production. Identification o osteoclast-committed precursors in vivo demonstrated that osteoblasts are involved in the distribution o osteoclast precursors in bone. Interleukin 34(IL-34)a novel ligand for c-Fms,plays a pivotal role in maintaining the splenic reservoir of osteoclast-committed precursors in M-CSF deficient mice. IL-34 is also able to act as a substitute for osteoblast-producing M-CSF in osteoclastogenesis. Wnt5 a,produced by osteoblasts,enhances osteoclast differentiation by upregulating RANK expression through activation of the noncanonical Wnt pathway. Semaphorin 3A produced by osteoblasts inhibits RANKL-induced osteoclast differentiation through the suppression of immunoreceptortyrosine-based activation motif signals. Thus,recent findings show that osteoclast differentiation is tightly regulated by osteoblasts through several different mechanisms. These newly identified molecules are expected to be promising targets of therapeutic agents in bone-related diseases.

Osteoblast Behavior on Hierarchical Micro-/Nano-Structured Titanium Surface

In the present work, osteoblast behavior on a hierarchical micro-/nano-structured titanium surface was investigated. A hi- erarchical hybrid micro-/nano-structured titanium surface topography was produced via Electrolytic Etching （EE）. MG-63 cells were cultured on disks for 2 h to 7 days. The osteoblast response to the hierarchical hybrid micro-/nano-structured titanium surface was evaluated through the osteoblast cell morphology, attachment and proliferation. For comparison, MG-63 cells were also cultured on Sandblasted and Acid-etched （SEA） as well as Machined （M） surfaces respectively. The results show signifi- cant differences in the adhesion rates and proliferation levels of MG-63 cells on EE, SLA, and M surfaces. Both adhesion rate and proliferation level on EE surface are higher than those on SLA and M surfaces. Therefore, we may expect that, comparing with SLA and M surfaces, bone growth on EE surface could be accelerated and bone formation could be promoted at an early stage, which could be applied in the clinical practices for immediate and early-stage loadings.

Interaction between Schwann Cells and Osteoblasts In Vitro

Aim Given the well-known properties of Schwann cells in promoting nerve regeneration, transplanting Schwann cells into implant sockets might be an effective method to promote sensory responses of osseointegrated implants. The aim of this study was to evaluate the interaction between Schwann cells and osteoblasts. Methodology Schwann cells derived from the sciatic nerves of neonatal rat were co-culured with osteoblasts using Transwell inserts. The proliferation of Schwann cells in the co-culture system was evaluated using methylthiazol tetrazolium （MTT） colorimetric method. Moreover, the secretions and mRNA levels of brain-derived neurotrophic factor （BDNF） and nerve growth factor （NGF） were measured by enzyme-linked immunosorbent assay （ELISA） and quantitative real-time PCR, respectively. In order to test the effect of Schwann cells on osteoblasts, alkaline phosphatase （ALP） staining and Alizerin red staining were performed as well. Results Schwann cells, which were co-cultured with the osteoblasts, showed an intact proliferation during the observation period. Moreover, the gene expression and synthesis of BDNF and NGF were not impaired by the osteoblasts. Meanwhile, co-cultured osteoblasts exhibited a significant increase in the proliferation on day 3 and 6 （P〈 0.05）. Co-culture of these two types of cells also led to a more intense staining of ALP and an elevated number of calcified nodules. Conclusion These findings demonstrate that, in the in vitro indirect co-culture environment, Schwann cells can maintain their normal ability to synthesize neurotrophins, which then enhance the proliferation and differentiation of osteoblasts.

Three-dimensional Expansion:In Suspension Culture of SD Rat's Osteoblasts in a Rotating Wall Vessel Bioreactor

Objective To study large-scale expansion of SD （Sprague-Dawley） rat＇s osteoblasts in suspension culture in a rotating wall vessel bioreactor （RWVB）. Methods The bioreactor rotation speeds were adjusted in the range of 0 to 20 rpm, which could provide low shear on the rnicrocarriers around 1 dyn/cm^2. The cells were isolated via sequential digestions of neonatal （less than 3 days old） SD rat calvaria. After the primary culture and several passages, the cells were seeded onto the microcarriers and cultivated in T-flask, spinner flask and RWVB respectively. During the culture period, the cells were counted and observed under the inverted microscope for morphology every 12 h. After 7 days, the cells were evaluated with scanning electron microscope （SEM） for histological examination of the aggregates. Also, the hematoxylin-eosin （HE） staining and alkaline phosphatase （ALP） staining were performed. Moreover, von-Kossa staining and Alizarin Red S staining were carded out for mineralized nodule formation. Results The results showed that in RWVB, the cells could be expanded by more than ten times and they presented better morphology and vitality and stronger ability to form bones. Conclusions The developed RWVB can provide the culture environment with a relatively low shear force and necessary three-dimensional （3D） interactions among cells and is suitable for osteopath expansion in vitro.

Effects of Lanthanum on Bone Resorbing Activity of Rabbit Mature Osteoclasts Co-Cultured with Osteoblasts

The effects of lanthanum （Ⅲ） on the bone resorbing activity of rabbit mature osteoclasts （OCs） in the presence of osteoblasts （OBs） were studied in vitro by measuring the number and area of absorption pits. La（ Ⅲ ） at concentrations ranging from 1.00 × 10^-5 to 1.00 × 10^-8 mol·L^-1 show no effect on mature OC number （P 〉 0.05）. In the OC-OB coculture systems without La（Ⅲ ）, osteoblasts alone did not influence the pit number and area whether the two kinds of cells were in contact or not （ P 〉 0.05）. Under the OC-OB not-in-contact condition, the effect of La（ Ⅲ ） on the bone-resorbing activity of OCs was similar to that of La（Ⅲ） in the absence of OBs （P 〉 0.05）. However, while OCs were in direct contact with OBs, the inhibitory effects of La（ Ⅲ ） on OCs＇ bone-resorbing activity decreased at the concentrations of 1.00 × 10^-5, 1.00×10^-6 and 1.00×10^-7mol·L^-1, and the promotion effects increased at 1.00×10^-8mol·L^-1 （P 〈0.05）. The results suggest that direct cell-cell contact between OC and OB be essential for OBs to play their role in regulating the response of OCs to La（ Ⅲ ）.

Cloning and Expression of Rat Transforming Growth Factor β1 cDNA in Osteoblasts

Summary: Rat transforming growth factor β1 (rTGFβ1) cDNA from rat lymphocytes was cloned by RT-PCR and inserted into pcDNA3 to construct an eukaryotic expression vector, which was named pcDNA3-TGFβ1. The cloned gene was confirmed to code rat TGFβ1 by restriction enzyme analysis. pcDNA3-TGFβ1 plasmid was transfected into rat osteoblasts by using liposome-mediated gene transfer technique and the expression of TGFβ1 was detected by using irnmunohistochemical staining assay. It was found that the rat TGFβ1 expression product was obviously detectable in the transfected osteoblasts in 48 h. High expression of TGFβ1 was obtained in the rat osteoblasts in which the constructed TGFβ1 expression vector was transfected.

Increased Expression of Receptor Activator of Nuclear Factor-κB Ligand in Osteoblasts from Adolescent Idiopathic Scoliosis Patients with Low Bone Mineral Density

Persistent generalized low bone mineral density (BMD) has been reported in patients with adolescent idiopathic scoliosis (AIS).However,the exact mechanisms and causes of the low BMD in AIS patients are largely unknown.The purpose of this study was to examine the relationship between the receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) levels in osteoblasts (OBs) from AIS patients with low BMD and with comparison made between the patients and controls.Twenty AIS patients and eight age-matched controls were included in the present study.The BMD of lumbar spine and proximal femur was measured in all subjects.OBs from the cancellous bone of each subject was harvested and primarily cultured.The mRNA and protein expression of RANKL and OPG in OBs was detected by RT-PCR and Western blotting.The results showed BMD was lower in AIS patients than in controls.A significantly higher mRNA and protein expression of RANKL was observed in OBs from AIS patients,while no significant difference was found in the expression of OPG between AIS patients and controls.As a result,RANKL/OPG ratio in patients with AIS was remarkably higher than controls.Our study preliminarily demonstrated expression of RANKL was higher in OBs from AIS patients with low BMD as compared with controls,suggesting the unbalanced RANKL/OPG ratio caused by an over-expression of RANKL in OBs may be responsible for the low BMD in AIS patients.

miR-23a/b regulates the balance between osteoblast and adipocyte differentiation in bone marrow mesenchymal stem cells

Age-related osteoporosis is associated with the reduced capacity of bone marrow mesenchymal stem cells （BMSCs） to differentiate into osteoblasts instead of adipocytes. However, the molecular mechanisms that decide the fate of BMSCs remain unclear. In our study, microRNA-23a, and microRNA-23b （miR-23a/b） were found to be markedly downregulated in BMSCs of aged mice and humans. The overexpression of miR-23a/b in BMSCs promoted osteogenic differentiation, whereas the inhibition of miR-23a/b increased adipogenic differentiation. Transmembrane protein 64 （Tmem64）, which has expression levels inversely related to those of miR-23a/b in aged and young mice, was identified as a major target of miR-23a/b during BMSC differentiation. In conclusion, our study suggests that miR-23a/b has a critical role in the regulation of mesenchymal lineage differentiation through the suppression of Tmem64.

Effects of Icariin on Expression of OPN mRNA and Type ⅠCollagen in Rat Osteoblasts in Vitro

To study the effects of Icariin on expression of osteopontin （OPN） mRNA and type Ⅰ collagen in rat osteoblasts in vitro and to explore its possible mechanisms in preventing osteoporosis. OB was isolated from calvaria of new-born new-born fetal Sprague-Dawley （SD） rats by means of modified sequential collagenase digestion and incubated in MEM medium and the cell morphology was observed under inverted phase contrast microscope, OB was identified by alkaline phosphatase （ALP） staining. Different concentration （0.1μg/mL, 1.0 μg/mL, 10 μ/mL） of Icariin was added to the OB and incubated. The effect of Icariin on the proliferation and osteogenesis of OB was monitored by MTT analysis. The expression of type l collagen was estimated with immunohistochemistry techniques. The expression levels of mRNA of OPN in the cells in every group were examined by reverse-transcriptase ploymerase chain reaction （RT-PCR）. The expression of OPN mRNA and type Ⅰ collagen was strengthened gradually with the increase of Icariin concentration and peaked with 10 μg/mL Icariin on the 5th day. Icariin could significantly promote the expression of OPN mRNA and type Ⅰ collagen in rat osteoblasts in vitro. The levels of expression of OPN mRNA and type Ⅰ collagen were changed with different concentration of Icariin. Icariin could effectively prevent and treat osteoporosis and promote the bone formation.

Osteoblast-restricted Disruption of the Growth Hormone Receptor in Mice Results in Sexually Dimorphic Skeletal Phenotypes

Growth hormone （GH） exerts profound anabolic actions during postnatal skeletal development, in part, through stimulating the production of insulin-like growth factor-1 （IGF-1） in liver and skeletal tissues. To examine the requirement for the GH receptor （GHR） in osteoblast function in bone, we used Cre-LoxP methods to disrupt the GHR from osteoblasts, both in vitro and in vivo. Disruption of GHR from primary calvarial osteoblasts in vitro abolished GH-induced signaling, as assessed by JAK2/STAT5 phosphorylation, and abrogated GH-induced proliferative and anti-apoptotic actions. Osteoblasts lacking GHR exhibited reduced IGF-l-induced Erk and Akt phosphorylation and attenuated IGF-1-induced proliferation and anti-apoptotic action. In addition, differentiation was modestly impaired in osteoblasts lacking GHR, as demonstrated by reduced alkaline phosphatase staining and calcium deposition. In order to determine the requirement for the GHR in bone in vivo, we generated mice lacking the GHR specifically in osteoblasts （△GHR）, which were born at the expected Mendelian frequency, had a normal life span and were of normal size. Three week-old, female AGHR mice had significantly reduced osteoblast numbers, consistent with the in vitro data. By six weeks of age however, female AGHR mice demonstrated a marked increase in osteoblasts, although mineralization was impaired; a phenotype similar to that observed previously in mice lacking IGF-1R specifically in osteoblasts. The most striking phenotype occurred in male mice however, where disruption of the GHR from osteoblasts resulted in a ＂feminization＂ of bone geometry in 16 week-old mice, as observed by faCT. These results demonstrate that the GHR is required for normal postnatal bone development in both sexes. GH appears to serve a primary function in modulating local IGF-1 action. However, the changes in bone geometry observed in male AGHR mice suggest that, in addition to facilitating IGF-1 action, GH may function to a greater extent than previously appreciated in establishing the sexual dimorphism of the skeleton.