Crosstalk between Wnt and bone morphogenetic protein signaling during osteogenic differentiati

Mesenchymal stem cells(MSCs)originate from many sources,including the bone marrow and adipose tissue,and differentiate into various cell types,such as osteoblasts and adipocytes.Recent studies on MSCs have revealed that many transcription factors and signaling pathways control osteogenic development.Osteogenesis is the process by which new bones are formed;it also aids in bone remodeling.Wnt/β-catenin and bone morphogenetic protein(BMP)signaling pathways are involved in many cellular processes and considered to be essential for life.Wnt/β-catenin and BMPs are important for bone formation in mammalian development and various regulatory activities in the body.Recent studies have indicated that these two signaling pathways contribute to osteogenic differen-tiation.Active Wnt signaling pathway promotes osteogenesis by activating the downstream targets of the BMP signaling pathway.Here,we briefly review the molecular processes underlying the crosstalk between these two pathways and explain their participation in osteogenic differentiation,emphasizing the canonical pathways.This review also discusses the crosstalk mechanisms of Wnt/BMP signaling with Notch-and extracellular-regulated kinases in osteogenic differentiation and bone development.

Low-intensity pulsed ultrasound reduces alveolar bone resorption during orthodontic treatment via Lamin A/C-Yes-associated protein axis in stem cells

BACKGROUND The bone remodeling during orthodontic treatment for malocclusion often requires a long duration of around two to three years,which also may lead to some complications such as alveolar bone resorption or tooth root resorption.Low-intensity pulsed ultrasound(LIPUS),a noninvasive physical therapy,has been shown to promote bone fracture healing.It is also reported that LIPUS could reduce the duration of orthodontic treatment;however,how LIPUS regulates the bone metabolism during the orthodontic treatment process is still unclear.AIM To investigate the effects of LIPUS on bone remodeling in an orthodontic tooth movement(OTM)model and explore the underlying mechanisms.METHODS A rat model of OTM was established,and alveolar bone remodeling and tooth movement rate were evaluated via micro-computed tomography and staining of tissue sections.In vitro,human bone marrow mesenchymal stem cells(hBMSCs)were isolated to detect their osteogenic differentiation potential under compression and LIPUS stimulation by quantitative reverse transcription-polymerase chain reaction,Western blot,alkaline phosphatase(ALP)staining,and Alizarin red staining.The expression of Yes-associated protein(YAP1),the actin cytoskeleton,and the Lamin A/C nucleoskeleton were detected with or without YAP1 small interfering RNA(siRNA)application via immunofluorescence.RESULTS The force treatment inhibited the osteogenic differentiation potential of hBMSCs;moreover,the expression of osteogenesis markers,such as type 1 collagen(COL1),runt-related transcription factor 2,ALP,and osteocalcin(OCN),decreased.LIPUS could rescue the osteogenic differentiation of hBMSCs with increased expression of osteogenic marker inhibited by force.Mechanically,the expression of LaminA/C,F-actin,and YAP1 was downregulated after force treatment,which could be rescued by LIPUS.Moreover,the osteogenic differentiation of hBMSCs increased by LIPUS could be attenuated by YAP siRNA treatment.Consistently,LIPUS increased alveolar bone density and decreased vertical bone absorption in vivo.The decreased expression of COL1,OCN,and YAP1 on the compression side of the alveolar bone was partially rescued by LIPUS.CONCLUSION LIPUS can accelerate tooth movement and reduce alveolar bone resorption by modulating the cytoskeleton-Lamin A/C-YAP axis,which may be a promising strategy to reduce the orthodontic treatment process.

AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

Use of bone morphogenetic proteins in mesenchymal stemcell stimulation of cartilage and bone repair

The extracellular matrix-associated bone morphogenetic proteins(BMPs) govern a plethora of biological processes. The BMPs are members of the transforming growth factor-β protein superfamily, and they actively participate to kidney development, digit and limb formation, angiogenesis, tissue fibrosis and tumor development. Since their discovery, they have attracted attention for their fascinating perspectives in the regenerative medicine and tissue engineering fields. BMPs have been employed in many preclinical and clinical studies exploring their chondrogenic or osteoinductive potential in several animal model defects and in human diseases. During years of research in particular two BMPs, BMP2 and BMP7 have gained the podium for their use in the treatment of various cartilage and bone defects. In particular they have been recently approved for employment in non-union fractures as adjunct therapies. On the other hand, thanks to their potentialities in biomedical applications, there is a growing interest in studying the biology of mesenchymal stem cell(MSC), the rules underneath their differentiation abilities, and to test their true abilities in tissue engineering. In fact, the specific differentiation of MSCs into targeted celltype lineages for transplantation is a primary goal of the regenerative medicine. This review provides an overview on the current knowledge of BMP roles and signaling in MSC biology and differentiation capacities. In particular the article focuses on the potential clinical use of BMPs and MSCs concomitantly, in cartilage and bone tissue repair.

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.

Induction of Bone Matrix Protein Expression by Native Bone Matrix Proteins in C2C12 Culture

Objective To study the expression of bone matrix protein （BMP） induced by bovine bone morphogenetic proteins （BMPs） in vitro. Methods Type 1 collagen, osteopontin （OPN）, osteonectin （ON）, osteocalcin （OC）, and bone sialoprotein （BSP） were detected by immunohistochemistry in C2C12 cultured from day 1 to day 28. Results The signaling of bone matrix protein expression became weaker except for type I collagen, OC and BSP after 5 days. Fourteen days after culture, the positive signaling of type I collagen, OPN, ON, OC, and BSP was gradually declined, and could be detected significantly as compared with that of the negative control on day 28. BMP assay showed that the lkaline phosphatase （ALP） activity was higher in C2C12 culture than in the control during the 14-day culture. Also, total protein and DNA significantly increased during the 14-day culture. High levels of ALP were seen in preosteoblasts and osteoblsts in vivo and in differentiating osteoblasts in vitro. ALP was well recognized as a marker reflecting osteoblastic activity. Conclusion Native bovine BMP induces conversion of myoblasts into osteoblasts, produces type I collagen, and plays significantly role in osteoinduction and bone matrix mineralization of C2C 12 in vitro.

Effect of transforming growth factor beta and bone morphogenetic proteins on rat hepatic stellate cell proliferation and transdifferentiation

AIM: To explore different roles of TGF-β (transforming growth factor beta) and bone morphogenetic proteins (BMPs)in hepatic stellate cell proliferation and trans-differentiation.METHODS: Hepatic stellate cells were isolated from male Sprague-Dawley rats. Sub-cultured hepatic stellate cells were employed for cell proliferation assay with WST-1 reagent and Western blot analysis with antibody against smooth muscle alpha actin (SMA).RESULTS: The results indicated that TGF-β1 significantly inhibited cell proliferation at concentration as low as 0.1 ng/ml, but both BMP-2 and BMP-4 did not affect cell proliferation at concentration as high as 10 ng/ml. The effect on hepatic stellate cell trans-differentiation was similar between TGFβ1 and BMPs. However, BMPs was more potent at transdifferentiation of hepatic stellate cells than TGF-β1. In addition, we observed that TGF-β1 transient reduced the abundance of SMA in hepatic stellate cells.CONCLUSION: TGF-β may be more important in regulation of hepatic stellate cell proliferation while BMPs may be the major cytokines regulating hepatic stellate cell transdifferentiation.

Bone morphogenetic proteins:Relationship between molecular structure and their osteogenic activity

Bone morphogenetic proteins(BMPs)are a family of potent,multifunctional growth factors belonging to transforming growth factor-(TGF-).They are highly conservative in structures.Over 20 members of BMPs with varying functions such as embryogenesis,skeletal formation,hematopoiesis and neurogenesis have been identified in human body.BMPs are unique growth factors that can induce the formation of bone tissue individually.BMPs can induce the differentiation of bone marrow mesenchymal stem cells into osteoblastic lineage and promote the proliferation of osteoblasts and chondrocytes.BMPs stimulate the target cells by specific membrane-bound receptors and signal transduced through mothers against decapentaplegic(Smads)and mitogen activated protein kinase(MAPK)pathways.It has been demonstrated that BMP-2,BMP-4,BMP-6,BMP-7,and BMP-9 play an important role in bone formation.This article focuses on the molecular characterization of BMPs family members,mechanism of osteogenesis promotion,related signal pathways of osteogenic function,relationships between structure and osteogenetic activity,and the interactions among family members at bone formation.

Bone Morphogenetic Proteins and Bone Metabolism

Bone morphogenetic proteins （BMPs）, a member of transforming growth factor-beta （TGF-beta） superfamily, can induce formation of cartilage and bone and also regulate osteoblasts, osteogenic genes and osteogenesis. BMPs together with hormones and local signals in cellular microenvironment determine differentiation of mesenchymal stem cells. Further researches about BMPs-related genes will provide a new way to un- derstand and treat metabolic bone diseases in humans and animals. This paper introduces bone metabolism-related BMPs and their transcription factors.

Cloning of Rabbit Bone Morphogenetic Protein 15 and Its Expression During in vitro Maturation of Rabbit Oocytes

Partial cDNA sequence of rabbit BMP15 was cloned by RT-PCR from rabbit ovaries, showing a similarity of 83%-90% with the BMP15 nucleotide sequences in humans, mice, ovine, sheep, cows and pigs. The expression of BMP15 in rabbit cumulus-oocyte complexs during oocytes in vitro maturation （IVM） was measured by fluorescent quantitative RT-PCR method. BMP 15 was expressed at low levels in immature oocytes and increased to the highest level at 16h of IVM, which coincides with the time of cumulus cell expansion, then declined slowly under IVM cultivation. The expression pattern of BMP 15 suggested that it might be important in cumulus expansion in rabbits.

Extraction, Purification and Identification of Bone Morphogenetic Protein in Conditioned Medium of Osteosarcoma Cell (MG-63)

Objective: To find out a method of extraction and purification of bone morphogenetic protein (BMP) from osteosarcoma cell conditioned medium, and evaluate the biological activity of BMP.Methods: Conditioned medium of osteosarcoma cell lines (MG-63) was collected, concentrated and dialyzed. The concentrated protein was purified through gel chromatography on Sephcryl-S-100. The purified protein was tested by BMP monoclonal antibody (McAb), its molecular weight (MW) was determined by SDS-PAGE and its biological activity was demonstrated by heterotopic ossification.Results: The purified protein was proved to be BMP by BMP McAb, had a satisfactory heterotopic ossification, and its MW was about 21 kD.Conclusion: BMP existed in the conditioned medium of osteosarcoma cell and had a satisfactory biological activity after purification. Because osteosarcoma cell can be cultured and grew for a long timein vitro, this method will be helpful to a vast extraction of BMP and clinical application. Key words osteosarcoma cell - conditioned medium - bone morphogenetic protein - protein purification This project was a key scientific and technological program of Hubei Provicial Scientific and Technological Committee (No. 002p1503).

Bone morphogenetic protein-7 represses hepatic stellate cell activation and liver fibrosis via regulation of TGF-β/Smad signaling pathway

BACKGROUND Liver fibrosis is a refractory disease whose persistence can eventually induce cirrhosis or even liver cancer.Early liver fibrosis is reversible by intervention.As a member of the transforming growth factor-beta(TGF-β)superfamily,bone morphogenetic protein 7(BMP7)has anti-liver fibrosis functions.However,little is known about BMP7 expression changes and its potential regulatory mechanism as well as the relationship between BMP7 and TGF-βduring liver fibrosis.In addition,the mechanism underlying the anti-liver fibrosis function of BMP7 needs to be further explored.AIM To investigate changes in the dynamic expression of BMP7 during liver fibrosis,interactions between BMP7 and TGF-β1,and possible mechanisms underlying the anti-liver fibrosis function of BMP7.METHODS Changes in BMP7 expression during liver fibrosis and the interaction between BMP7 and TGF-β1 in mice were observed.Exogenous BMP7 was used to treat mouse primary hepatic stellate cells(HSCs)to observe its effect on activation,migration,and proliferation of HSCs and explore the possible mechanism underlying the anti-liver fibrosis function of BMP7.Mice with liver fibrosis received exogenous BMP7 intervention to observe improvement of liver fibrosis by using Masson’s trichrome staining and detecting the expression of the HSC activation indicator alpha-smooth muscle actin(α-SMA)and the collagen formation associated protein type I collagen(Col I).Changes in the dynamic expression of BMP7 during liver fibrosis in the human body were further observed.RESULTS In the process of liver fibrosis induced by carbon tetrachloride(CCl4)in mice,BMP7 protein expression first increased,followed by a decrease;there was a similar trend in the human body.This process was accompanied by a sustained increase in TGF-β1 protein expression.In vitro experiment results showed that TGF-β1 inhibited BMP7 expression in a time-and dose-dependent manner.In contrast,high doses of exogenous BMP7 inhibited TGF-β1-induced activation,migration,and proliferation of HSCs;this inhibitory effect was associated with upregulation of pSmad1/5/8 and downregulation of phosphorylation of Smad3 and p38 by BMP7.In vivo experiment results showed that exogenous BMP7 improved liver fibrosis in mice.CONCLUSION During liver fibrosis,BMP7 protein expression first increases and then decreases.This changing trend is associated with inhibition of BMP7 expression by sustained upregulation of TGF-β1 in a time-and dose-dependent manner.Exogenous BMP7 could selectively regulate TGF-β/Smad pathway-associated factors to inhibit activation,migration,and proliferation of HSCs and exert antiliver fibrosis functions.Exogenous BMP7 has the potential to be used as an antiliver fibrosis drug.

Effect of Danshao Huaxian capsule on Gremlin and bone morphogenetic protein-7 expression in hepatic fibrosis in rats

AIM: To observe the effect of Danshao Huaxian capsule (DHC) on the expression of Gremlin and bone morphogenetic protein-7 (BMP-7) in the liver of hepatic fibrosis rats.

Growth-associated protein 43 and neural cell adhesion molecule expression following bone marrow-derived mesenchymal stem cell transplantation in a rat model of ischemic brain injury

BACKGROUND： Transplantation of bone marrow-derived mesenchymal stem cells （BMSCs） improves motor functional recovery, but the mechanisms remain unclear. OBJECTIVE： To investigate expression of growth-associated protein 43 （GAP-43） and neural cell adhesion molecule following BMSC transplantation to the lateral ventricle in rats with acute focal cerebral ischemic brain damage. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment using immunohistochemistry was performed at the laboratories of Department of Neurology, Renmin Hospital of Wuhan University and Doctoral Scientific Research Work Station of C-BONS PHARMA, Hubei Province, China, from January 2007 to December 2008. MATERIALS： Monoclonal mouse anti-rat 5-bromo-2-deoxyuridine and neural cell adhesion molecule antibodies were purchased from Sigma, USA; monoclonal mouse anti-rat GAP-43 antibody was purchased from Wuhan Boster, China. METHODS： Rat models of right middle cerebral artery occlusion were established using the thread method. At 1 day after middle cerebral artery occlusion, 20μL culture solution, containing 5×10^5 BMSCs, was transplanted to the left lateral ventricle using micro-injection. MAIN OUTCOME MEASURES： Scores of neurological impairment were measured to assess neural function. Expression of GAP-43 and neural cell adhesion molecule at the lesion areas was examined by immunohistochemistry. RESULTS： GAP-43 and neural cell adhesion molecule expression was low in brain tissues of the sham-operated group, but expression increased at the ischemic boundary （P 〈 0.05）. Transplantation of BMSCs further enhanced expression of GAP-43 and neural cell adhesion molecule （P 〈 0.05） and remarkably improved neurological impairment of ischemic rats （P 〈 0.05）. CONCLUSION： BMSC transplantation promoted neurological recovery in rats by upregulating expression of GAP-43 and neural cell adhesion molecule.

Transfection of bone marrow mesenchymal stem cells using green fluorescence protein labeled hVEGF165 recombinant plasmid mediated by liposome

Objective:To study the role of bone marrow mesenchymal stem cells(BMSCs)in construction of vascularized engineered tissue.Methods:hVEGF165 was amplified via RT-PCR before recombinant with pShuttle-green fluorescence protein;green fluorescent protein(GFP)-CMV.Then the recombinant shuttle plasmid was transfected into BMSCs with Lipofectamine^(TM)2000 for packaging and amplifying.hVTGF165 mRNA expression in BMSCs cells was tested.Results:The sequence of hVEGFI65 in pShutlle-GFP-hVFGF165 plasmid was confirimed by double-enzyme cleavage method and sequencing.hVECF165 was highly expressed in BMSCs.Conclusions:The GFP/hVECF165 recombinant plasmid vector was constructed successfully and expressed effectively in host cells,which may be helpful for discussing the possibility of the application of VEGF165-BMSCs in tissue engineering and ischemic disease cure.

Heterotopic ossification after the use of recombinant human bone morphogenetic protein-7

AIM To present the incidence of heterotopic ossification after the use of recombinant human bone morphogenetic protein-7(rhB MP-7) for the treatment of nonunions.METHODS Bone morphogenetic proteins(BMPs) promote bone formation by auto-induction. Recombinant human BMP-7 in combination with bone grafts was used in 84 patients for the treatment of long bone nonunions. All patients were evaluated radiographicaly for the development of heterotopic ossification during the standard assessment for the nonunion healing. In all patients(80.9%) with radiographic signs of heterotopic ossification, a CT scan was performed. Nonunion site palpation and ROM evaluation of the adjacent jointswere also carried out. Factors related to the patient(age, gender), the nonunion(location, size, chronicity, number of previous procedures, infection, surrounding tissues condition) and the surgical procedure(graft and fixation type, amount of rhB MP-7) were correlated with the development of heterotopic ossification and statistical analysis with Pearsons χ~2 test was performed.RESULTS Eighty point nine percent of the nonunions treated with rh BMP-7, healed with no need for further procedures. Heterotopic bone formation occurred in 15 of 84 patients(17.8%) and it was apparent in the routine radiologi-cal evaluation of the nonunion site, in a mean time of 5.5 mo after the rh BMP-7 application(range 3-12). The heterotopic ossification was located at the femur in 8 cases, at the tibia in 6, and at the humerus in οne patient. In 4 patients a palpable mass was present and only in one patient, with a para-articular knee nonunion treated with rhB MP-7, the size of heterotopic ossification affected the knee range of motion. All the patients with heterotopic ossification were male. Statistical analysis proved that patient's gender was the only important factor for the development of heterotopic ossification(P = 0.007). CONCLUSION Heterotopic ossification after the use of rh BMP-7 in nonunions was common but it did not compromise the final clinical outcome in most cases, and affected only male patients.

Expression of Vascular Endothelial Growth Factor in Bone Morphogenetic Protein-2 Induced Osteogenesis

An experimental model of femoral muscular pouch in 20 mice was adopted. The expression of VEGF was examined by in situ hybridization method and immunohistochemical method in bone morphogenetic protein- 2 induced osteogenesis . The experimental results demonstrated that the expression signals of VEGF mRNA and VEGF appeared in cytoplasm during condensation of mesenehymal cell. As the mesenchymal cells differentiated into precartilage, the expression signals decreased in mesenehymal cells, but increased in chondrocytes and kept getting denser in the process of cartilage maturity. The peak expression of VEGF mRNA and VEGF in the experimental group appeared on the 14 th day, accompanied by numerons hypertrophic chondrocytes. When mature cartilage calcified and neu, bone trabecula formed, the expression of VEGF mRNA and VEGF decreased in chondrocytes, but still expressed moderately in the osteoblasts and osteocytes. Signals of VEGF mRNA and VEGF can not be detected in the control groups.

Adipokines and C-reactive protein in relation to bone mineralization in pediatric nonalcoholic fatty liver disease

AIM: To investigate bone mineral density (BMD) in obese children with and without nonalcoholic fatty liver disease (NAFLD); and the association between BMD and serum adipokines, and high-sensitivity C-reactive protein (HSCRP). METHODS: A case-control study was performed. Cases were 44 obese children with NAFLD. The diagnosis of NAFLD was based on magnetic resonance imaging (MRI) with high hepatic fat fraction (≥ 5%). Other causes of chronic liver disease were ruled out. Controls were selected from obese children with normal levels of aminotransferases, and without MRI evidence of fatty liver as well as of other causes of chronic liver diseases. Controls were matched (1-to 1-basis) with thecases on age, gender, pubertal stage and as closely as possible on body mass index-SD score. All participants underwent clinical examination, laboratory tests, and whole body (WB) and lumbar spine (LS) BMD by dual energy X-ray absorptiometry. BMDZ-scores were calcu- lated using race and gender specific LMS curves. RESULTS: Obese children with NAFLD had a significantly lower LS BMDZ-score than those without NAFLD [mean, 0.55 (95%CI: 0.23-0.86) vs 1.29 (95%CI: 0.95-1.63); P < 0.01]. WB BMD Z-score was also decreased in obese children with NAFLD compared to obese children with no NAFLD, though borderline significance was observed [1.55 (95%CI: 1.23-1.87) vs 1.95 (95%CI: 1.67-2.10); P = 0.06]. Children with NAFLD had significantly higher HSCRP, lower adiponectin, but similar leptin levels. Thirty five of the 44 children with MRI-diagnosed NAFLD underwent liver biopsy. Among the children with biopsy-proven NAFLD, 20 (57%) had nonalcoholic steatohepatitis (NASH), while 15 (43%) no NASH. Compared to children without NASH, those with NASH had a significantly lower LS BMD Z-score [mean, 0.27 (95%CI: -0.17-0.71) vs 0.75 (95%CI: 0.13-1.39); P < 0.05] as well as a significantly lower WB BMD Z-score [1.38 (95%CI: 0.89-1.17) vs 1.93 (95%CI: 1.32-2.36); P < 0.05]. In multiple regression analysis, NASH (standardized β coefficient, -0.272; P < 0.01) and HSCRP (standardized β coefficient, -0.192; P < 0.05) were significantly and independently associated with LS BMD Z-score. Similar results were obtained when NAFLD (instead of NASH) was included in the model. WB BMD Z-scores were significantly and independently associated with NASH (standardized β coefficient, -0.248;P < 0.05) and fat mass (standardized β coefficient, -0.224;P < 0.05). CONCLUSION: This study reveals that NAFLD is associated with low BMD in obese children, and that systemic, low-grade inflammation may accelerate loss of bone mass in patients with NAFLD.

The biological function of type I receptors of bone morphogenetic protein in bone

Bone morphogenetic proteins （BMPs） have multiple roles in skeletal development, homeostasis and regeneration. BMPs signal via type I and type II serine/threonine kinase receptors （BMPRI and BMPRII）. In recent decades, genetic studies in humans and mice have demonstrated that perturbations in BMP signaling via BMPRI resulted in various diseases in bone, cartilage, and muscles. In this review, we focus on all three types of BMPRI, which consist of activin-like kinase 2 （ALK2, also called type IA activin receptor）, activin- llke kinase 3 （ALK3, also called BMPRIA）, and activin-like kinase 6 （ALK6, also called BMPRIB）. The research areas covered include the current progress regarding the roles of these receptors during myogenesis, chondrogenesis, and osteogenesis. Understanding the physiological and pathological functions of these receptors at the cellular and molecular levels will advance drug development and tissue regeneration for treating musculoskeletal diseases and bone defects in the future.

Cell multiplication, apoptosis and p-Akt protein expression of bone mesenchymal stem cells of rat under hypoxia environment

Objective ：To elucidate whether cell multiplication, apoptosis, glucose intake and p-Akt protein expression of bone Mesenchyreal Stem Cells（MSCs） of rats is influenced by a hypoxic environment ex vivo. Methods ：Passage 3 of bone marrow MSCs taken from Wistar rats,were cultured in a culturing chamber with 94%N2,1%O2,5%CO2 at 37℃. At different hypoxia time points ,0,0.5, 1,4 and 8 h, glucose uptake was assayed by using radiation isotope ^3H-G, Apoptotic Rate（AR） and dead rate（DR） were analyzed by flow cytometry（FCM） after Annexin V/PI staining, cell multiplication（by MTr methods） and p-Akt protein by immunocytochemistry and western blot. Results ：Assay for CD29^± ,CD44^± ,CD71^± ,CD34^-, Tn T^±（after 5-azacytidine agent inducing） and ALP^±（after bone differentiation agent inducing） suggested these bone-derived cells were MSCs. The ^3H-G intaking ratio （CPM/ flask value：157 ± 11,110 ± 11,107 ± 13,103 ± 10,100 ± 9 and 98 ± 10） of MSCs at different hypoxia time points, significantly decreased compared to that of normoxia（P 〈 0.01） and tended to descend slowly with hypoxia time duration, for which there was no statistical significance（P 〉 0.05）. The AR（0.09 ± 2.03%,12.9 ± 1.72%,13.7 ± 2.26%,13.8 ± 3.01%,14.1 ± 2.78% and 14.7 ± 4.01% at 0,0.5,1,4 and 8 h,respectively,P 〈 0.01） and DR （0.04, ± 1.79% ,0.93 ± 1.85% ,3.11 ± 2.14% ,4.09 ± 2.36% ,4.72 ± 2.05% and 4.91 ± 3.72% at 0,0.5,1,4 and 8 h, respectively, P 〈 0.05） at different hypoxia time points significantly increased compared to those time in normoxia; The AR further went up with time （P 〈 0.05）, however there was no statistical significance （P 〉 0.05） for the DR. Optical absorption value of MTr methods at different hypoxia time points significantly decreased compared to those with a corresponding normoxia time （P 〈 0.01） and degraded with time （in an hypoxic environment -P 〈 0.01）. IOD of p-Akt protein of MSCs at different hypoxia time points significantly increased （0.367 ± 0.031,0.556 ± 0.023,0.579 ± 0.013, 0.660 ± 0.024, 0.685 ± 0.039 and 0.685 ± 0.011, respectively） compared to their equivalents in normoxia （P〈0.05）, however, there was no statistical significance （P 〉 0.05） for different hypoxia time points. Hypoxia may result in ultramicrostructure changes, such as defluvium of Microvilli, apoptotic body, ＂margination＂ and so on and are further aggravated with hypoxia time stretching. Conclusion： Hypoxia may lead to a depression of MSCs intaldng glucose, creep of cell multiplication, upregulation of p-Akt protein and apoptosis of MSCs ex vivo.