RANKL expression is related to the differentiation state of human osteoblasts.

Human osteoblast phenotypes that support osteoclast differentiation and bone formation are not well characterized. Osteoblast differentiation markers were examined in relation to RANKL expression. RANKL expression was induced preferentially in immature cells. These results support an important link between diverse osteoblast functions. Cells of the osteoblast lineage support two apparently distinct functions: bone formation and promotion of osteoclast formation. The aim of this study was to examine the relationship between these phenotypes in human osteoblasts (NHBC), in terms of the pre-osteoblast marker, STRO-1, and the mature osteoblast marker, alkaline phosphatase (AP), and the expression of genes involved in osteoclast formation, RANKL and OPG. The osteotropic stimuli, 1alpha,25(OH)2vitamin D3 (vitD3) and dexamethasone, were found to have profound proliferative and phenotypic effects on NHBCs. VitD3 inhibited NHBC proliferation and increased the percentage of cells expressing STRO-1 over an extended culture period, implying that vitD3 promotes and maintains an immature osteogenic phenotype. Concomitantly, RANKL mRNA expression was upregulated and maintained in NHBC in response to vitD3. Dexamethasone progressively promoted the proliferation of AP-expressing cells, resulting in the overall maturation of the cultures. Dexamethasone had little effect on RANKL mRNA expression and downregulated OPG mRNA expression in a donor-dependent manner. Regression analysis showed that RANKL mRNA expression was associated negatively with the percentage of cells expressing AP (p < 0.01) in vitD3- and dexamethasone-treated NHBCs. In contrast, RANKL mRNA expression was associated positively with the percentage of STRO-1+ cells (p < 0.01). In NHBCs sorted by FACS based on STRO-1 expression (STRO-1bright and STRO-1dim populations), it was found that vitD3 upregulated the expression of RANKL mRNA preferentially in STRO-1bright cells. The results suggest that immature osteoblasts respond to osteotropic factors in a potentially pro-osteoclastogenic manner. Additionally, the dual roles of osteoblasts, in supporting osteoclastogenesis or forming bone, may be performed by the same lineage of cells at different stages of their maturation.     

Expression of preosteoblast markers and Cbfa-1 and Osterix gene transcripts in stromal tumour cells of giant cell tumour of bone

In giant cell tumour of bone (GCT), mononuclear stromal cells, which represent the neoplastic component of this lesion, regulate the formation of multinucleated osteoclast-like giant cells which are the characteristic hallmark of this tumour. However, the origin of stromal tumour cells has not yet been clearly defined. In this study, we evaluated several osteoblast markers including collagen type I, bone sialoprotein (BSP), osteonectin and osteocalcin in GCT using immunohistochemical techniques. Amongst the 13 GCT specimens and 7 GCT stromal cell (GCTSC) cultures studied, majority of the GCTSC synthesized type I collagen, BSP and osteonectin proteins but did not produce the differentiated osteoblast marker, osteocalcin. We further examined the regulation of several important osteogenic genes such as Cbfa-1, osterix and osteocalcin, and regulation of ALP activity in GCTSC in culture by bone morphogenetic protein 2 (BMP-2). Real-time PCR analysis indicated that Cbfa-1, osterix and osteocalcin mRNA were present in primary cultures of GCTSC. The addition of BMP-2 upregulated Cbfa-1 and osterix gene expression within 12 h and the enhancement was still observed at 24 h. ALP activity was minimal in untreated GCTSC in cultures. The number of ALP-positive GCTSC was significantly increased following treatment with BMP-2 or combinations with beta-glycerophosphate and ascorbic acid. In contrast, BMP enhancement of osterix mRNA level and ALP activity was also seen in SaOS2 osteoblast-like cells, but not in the primary culture of normal human skin fibroblasts. In summary, our data suggest that GCT stromal tumour cells may have an osteoblastic lineage and retain the ability to differentiate into osteoblasts.     

IGF-I does not affect the proliferation or early osteogenic differentiation of human marrow stromal cells

The ability of insulin-like growth factor-I (IGF-I) to regulate the proliferation and differentiation of primitive osteogenic precursors (CFU-F) has been investigated in cultures of bone marrow stromal cells (BMSC) derived from a large cohort of adult human donors. Treatment with IGF-I (0.1-20 ng/mL, days 0-28) had no consistent effect on the number or size of colonies that formed or the proportion of colonies that expressed the developmental marker alkaline phosphatase (AP). At the end of primary culture, similar numbers of cells were harvested from the control and IGF-I-treated groups and there was no detectable difference in the expression of AP (activity or percentage of positive cells) or the developmental marker STRO-1. This was found to be the case whether IGF-I was added alone or in combination with 10 nM dexamethasone (Dx), a known inducer of osteogenic differentiation in this cell culture system. In contrast, cells derived from the same cohort of donors responded to treatment with fibroblast growth factor-2 (FGF-2) with an increase in the number and size of the colonies that formed, in proliferation and in the number of cells recovered in STRO-1(+)/AP(+) (osteoprogenitor) fraction. Further analysis revealed that the majority of BMSC expressed the alpha and beta subunits of the type 1 receptor for IGF-I (IGF-IR), in the expected 1:1 ratio. Treatment with Dx did not affect the expression of these receptor subunits (percentage of positive cells or number of sites per cell) but did increase the proportion of cells present in the IGF-I(+)/AP(+) fraction. The results of this investigation suggest that the beneficial effects of IGF-I on the skeleton are not mediated primarily via an effect on osteoprogenitor fraction and are thus consistent with the hypothesis that the effects of IGF-I are differentiation dependent and restricted largely to the more mature cells of the osteoblast lineage.     

Inhibition of osteoblast differentiation but not adipocyte differentiation of mesenchymal stem cells by sera obtained from aged females

Aging is associated with decreased osteoblast-mediated bone formation leading to bone loss and increased risk for osteoporotic fractures. However, the cellular mechanisms responsible for impaired osteoblast functions are poorly understood. In the present study, we hypothesized that changes in bone microenvironment composition with aging are responsible for impaired osteoprogenitor cell recruitment and differentiation. As a model for bone microenvironment, we examined the effects of sera obtained from young (age 20-30 year old [yo], n=20) and old (70-84 yo, n=19) healthy female donors on cell proliferation and differentiation capacity into osteoblasts and adipocytes of human mesenchymal stem cells (hMSC). Cell proliferation rate determined by counting cell number was similar when the cells were cultured in the presence of media containing 5% sera from old or from young donors. Similarly, the number of adipocytes and levels of adipocytic gene expression was similar in cultures incubated with sera from young or old donors. We observed decreased osteoblastic gene expression in hMSC cultured either in pooled or individual sera of old donors compared to sera from young donors: core binding factor/runt-related binding factor 2 (Cbfa1/Runx2) 46%+/-2% (P<0.05), alkaline phosphatase (ALP) 45%+/-2% (P<0.05), collagen type I (Col-I) 50%+/-1% (P<0.05), and osteocalcin 65%+/-3% (P<0.05). This down-regulation of the mRNA was accompanied by reduced ALP enzyme activity by 25%+/-1% (P<0.01), immunocytochemical staining for osteoblastic markers: ALP, Col-I, and bone sialoprotein (BSP) as well as reduced in vitro mineralization as determined by Alizarin red staining. In conclusion, age-related changes in the serum composition and possibly hMSC microenvironment may contribute to the impaired osteoblast functions with aging. The factors mediating these changes remain to be determined.     

Bone sialoprotein enhances migration of bone marrow stromal cells through matrices by bridging MMP-2 to alpha(v)beta3-integrin.

BMSCs migrate through matrix barriers and differentiate into osteoblasts. BSP enhances osteogenic cell migration through basement membrane and collagen matrices in vitro by localizing MMP-2 on the cell surface through alpha(v)beta(3)-integrin. INTRODUCTION: The specific mechanisms by which bone marrow stromal cells (BMSCs) leave their primary sites, move through matrices encountered during homing to their site of final differentiation, and remove preexisting matrices in preparation for bone matrix production are not well understood. MATERIALS AND METHODS: The enhanced migration of human osteoblast precursor cells through matrix barriers by bone sialoprotein (BSP) was studied by a modified Boyden-chamber assay. The bridging of normally soluble matrix metalloproteinase 2 (MMP-2) to the cell surface receptor, alpha(v)beta(3)-integrin, by BSP was analyzed by flow cytometry. RESULTS: BSP enhanced the in vitro passage of BMSCs and pre-osteoblasts through matrix barriers (Matrigel and denatured type I collagen) in a dose-dependent manner. An intact ArgGlyAsp (RGD) was required in the BSP for enhanced migration through the barriers but was not sufficient, as shown by the inactivity of two other SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family members, osteopontin and dentin matrix protein-1. The specificity of the BSP enhancement activity was apparently caused by this molecule's ability to bridge MMP-2 to the cell surfaces. CONCLUSIONS: Pre-osteoblasts and their BMSC precursors may use MMP-2/BSP/integrin complexes to disrupt matrix barriers during migration to their final destinations in vivo.     

辛伐他汀对人牙髓干细胞成骨活性的影响

目的：研究辛伐他汀对人牙髓干细胞成骨活性的影响.方法：将体外分离培养的人牙髓干细胞分别接种于含有不同浓度辛伐他汀的矿化培养液中诱导培养,测定碱性磷酸酶活性及骨唾液酸蛋白基因的表达情况.结果：与对照组比较,适宜浓度辛伐他汀（1×10^-6mol/L、1×10-7mol/L、1×10-8mol/L）促进人牙髓干细胞的成骨活性作用更为明显,差异具有统计学意义（P＜0.05）,当辛伐他汀浓度为1×10-7mol/L时,促进作用最显著.结论：适宜浓度的辛伐他汀可以有效促进人牙髓干细胞的成骨活性.     

miR-187-5p 对骨髓间充质干细胞成骨分化能力的调控作用

目的利用小鼠骨髓间充质干细胞(BMSCs)的成骨分化模型,探讨miR-187-5p在成骨分化中的表达趋势及调控作用。方法通过切除雌性小鼠双侧卵巢构建小鼠骨质疏松模型;应用qRT-PCR技术检测组织和细胞中miR-187-5p的表达;应用基因转染技术观察过表达或敲减miR-187-5p对BMSCs向成骨分化的影响;应用茜素红和碱性磷酸酶染色检测BMSCs中矿化结节的数量和矿化区域的染色面积。结果 qRT-PCR结果显示miR-187-5p在骨质疏松模型小鼠的骨组织及BMSCs中均表达下降。过表达miR-187-5p可提高ALP、Collagen-1、Runx2、BMP4、OCN和OPN等成骨分化相关基因mRNA的表达,促进BMSCs向成骨分化;而敲减miR-187-5p降低ALP等成骨分化相关基因mRNA的表达,抑制BMSCs向成骨分化。在体实验同样证实,过表达miR-187-5p可以显著促进骨质疏松小鼠BMSCs向成骨分化,改善小鼠的骨质疏松表型。结论过表达miR-187-5p促进BMSCs向成骨分化,敲减miR-187-5p抑制BMSCs向成骨分化。     

Lycium barbarum polysaccharide protects against osteonecrosis of femoral head via regulating Runx2 expression

BackgroundOsteonecrosis of femoral head (ONFH) is a pathological state caused by lack of blood supply in femoral head. This study aimed to explore the function of Lycium barbarum polysaccharide (LBP), an antioxidant agent extracted from L. barbarum, on ONFH.MethodsOsteonecrosis rat model was generated using lipopolysaccharide (LPS) and methylprednisolone followed by examination of body weight, blood glucose, morphology, and BMSC osteoblast differentiation. The effect and underlying mechanism of LBP on the proliferation, apoptosis, and osteoblast differentiation of BMSC were determined with or without LPS or hypoxia treatment using CCK-8. Alizarin Red S staining, flow cytometry, and western blot, respectively.ResultLBP could protect against glucocorticoid-induced ONFH in rats, resulting in improved sparse trabecular bone, empty lacunae and bone cell coagulation. Moreover, LBP promoted the proliferation and osteoblast differentiation of bone mesenchymal-derived stem cells (BMSCs) in a dose-dependent manner. Furthermore, LBP enhanced osteoblast differentiation of BMSCs under hypoxia condition. Mechanistically, we found that LBP treatment enhanced Runx2 and ALP expression in BMSCs. LBP restored the expression of Runx2 and ALP under hypoxia, suggesting that LBP might be involved in regulating Runx2/ALP expression and contributed to osteoblast differentiation. Knockdown of Runx2 significantly inhibited BMSCs proliferation, while LBP treatment did not rescue the osteoblast differentiation ability of BMSCs with Runx2 knockdown.ConclusionOur findings suggested that LBP protects against ONFH via regulating Runx2 expression, which could be utilized to treat patients suffering ONFH.     

骨肉瘤成骨细胞表型与临床因素的相关性研究

目的采集手术切除未经培养的骨肉瘤标本,研究肿瘤中成骨细胞的分化及其与临床因素的关系。方法观察骨肉瘤中成骨细胞相关基因表达的频率、特异性,以及骨肉瘤成骨细胞表型与生物学行为的关系。应用RT-PCR方法研究48例骨肉瘤标本的碱性磷酸酶(ALP)、骨钙素(OC)和骨桥蛋白(ON)的mRNA转录。磷酸甘油酸激酶(PGK)被用于内参照。结果在大多数骨肉瘤标本中可以发现ALP或OC的mRNA转录(分别为93.75%,79.17%)。在所有的肉瘤检测中均可发现ON的mRNA转录。在骨肉瘤的成骨细胞亚型中可见到ALP和OC的高水平表达。肺转移瘤OC的mRNA表达弱于原发灶。肿瘤细胞有高水平OC表达的病例组有较高的生存率(P=0.045)。结论OC是目前最好的成骨细胞特异性标记物,将来可能用于检测骨肉瘤患者外周血中循环的肿瘤细胞。     

Identification of candidate genes involved in the reversal of malignant phenotype of osteosarcoma cells transfected with the liver/bone/kidney alkaline phosphatase gene

Alkaline phosphatases (ALPs) are a family of cell surface glycoproteins that catalyze the hydrolysis of phosphomonoesters with release of inorganic phosphate. Liver/bone/kidney (L/B/K) ALP participates in bone mineralization, but its other physiological and pathological functions remain obscure. In human osteosarcoma, an inverse relationship has been found between cellular L/B/K ALP expression and aggressiveness. To explore this relationship, we employed cDNA microarray technology to characterize and compare the gene expression profile of two U-2 OS osteosarcoma clones with high L/B/K ALP activity (U-2/ALP28 and U-2/ALP40) and one with contrasting characteristics (U-2/ALP23). We identified 79 differentially expressed genes (58 upregulated in U-2/ALP28 and U-2/ALP40 compared to U-2/ALP23). Using GenMAPP/MAPPFinder, we highlighted nine functional groups strictly related to high L/B/K ALP activity, including microtubule-based movement and cell adhesion groups, two functions well related to tumor invasiveness. Notably, cadherin 13 (CDH13) and caveolin 1 (CAV1) genes were upregulated in our cells. Since these two genes are involved in cell-cell adhesion and cell growth, their co-expression with L/B/K ALP could help explain the lower levels of malignancy found in osteosarcoma cells with high L/B/K ALP activity. Although functional studies are needed to better define the role of CDH13 and CAV1 in the malignant behavior of osteosarcoma cells, the data presented here provide an aid to understanding the biological functions of L/B/K ALP in bone tumors.     

骨形态发生蛋白-2对人脐带间充质干细胞增殖和分化的影响

目的 观察骨形态发生蛋白-2(BMP-2)对人脐带间充质干细胞(hUCMSCs)增殖和分化的影响.方法 体外培养hUCMSCs,在培养基中加入20 mg/L BMP-2,噻唑蓝(MTT)比色法观察BMP-2对hUCMSCs的增殖效果,流式细胞术检测BMP-2作用后细胞表面STRO-1的表达,逆转录-聚合酶链反应(RT-PCR)测量BMP-2作用后hUCMSCs骨桥蛋白(OPN)、碱性磷酸酶(ALP)、Ⅰ型胶原蛋白(COL1)的mRNA表达变化,碱性磷酸酶染色观察hUCMSCs在BMP-2培养基作用下ALP染色变化,Von kossa染色实验观察BMP-2对hUCMSCs钙结节的形成.结果 细胞在未加BMP-2和加BMP-2培养基中培养1、3、5、7 d,虽然细胞的增殖率上升,但各组间比较差异无统计学意义(P＞0.05),同时发现在2%血清的培养基中培养7 d后,hUCMSCs的增殖促进约10%左右.BMP-2培养7 d后细胞表面STRO-1阳性细胞比例上升明显,由25.1±4.0上升至51.1±6.4,差异有统计学意义(P＜0.01).BMP-2培养条件下COL1 mRNA的表达增强,差异有统计学意义(P＜0.05),OPN mRNA出现表达,ALP mRNA比无BMP-2培养明显增强,差异有统计学意义(P＜0.05).在BMP-2培养条件下ALP染色出现大片细胞阳性染色.在培养28 d,Von kossa染色出现明显的钙结节.结论 BMP-2对hUCMSCs成骨诱导分化作用明显,而增殖作用很弱.     

健骨颗粒含药血清调控miR-141对小鼠骨髓间充质干细胞成骨分化的影响

目的观察健骨颗粒含药血清对去卵巢模型小鼠BMSCs成骨分化的影响,并研究其调控miR-141影响BMSCs分化的可能机制。方法流式细胞计数法鉴定BMSCs;含药血清和空白血清分别干预体外培养的BMSCs;显微镜下观察细胞形态结构,碱性磷酸酶染色、定量检测,茜素红染色;RT-PCR检测miR-141及DLx5、Msx2、Runx2的基因表达情况;Western blot检测DLx5、Msx2及Runx2的蛋白表达情况。结果流式细胞计数法鉴定提取细胞符合实验要求;镜下观察发现两组细胞有差异,含药血清组细胞群落间结晶更为密集;ALP染色及定量、茜素红染色结果含药血清组均优于空白血清组;RT-PCR检测Dlx5、Msx2、Runx2及miR-141的基因表达结果:含药血清组miR-141和Msx2基因表达较空白血清组显著降低(P<0.01),而Dlx5、Runx2的基因表达显著升高(P<0.01);Western blot检测DLx5、Msx2、Runx2的蛋白表达结果:含药血清组较空白血清组相比,Msx2的蛋白表达略微下降(P<0.05),而Dlx5、Runx2的蛋白表达相对升高(P<0.01)。结论健骨颗粒可以调低miR-141的表达,进而调高Dlx5的表达,削弱Dlx5的同源异形基因Msx2对Runx2抑制作用,通过miR-141调控Dlx5/Msx2/Runx2信号通路促进BMSCs成骨分化达到预防和治疗绝经后骨质疏松的目的。     

Differentiation of human marrow stromal precursor cells: bone morphogenetic protein-2 increases OSF2/CBFA1, enhances osteoblast commitment, and inhibits late adipocyte maturation.

Because regulation of the differentiation to osteoblasts and adipocytes from a common progenitor in bone marrow stroma is poorly understood, we assessed effects of bone morphogenetic protein-2 (BMP-2) on a conditionally immortalized human marrow stromal cell line, hMS(2-6), which is capable of differentiation to either lineage. BMP-2 did not affect hMS(2-6) cell proliferation but enhanced osteoblast differentiation as assessed by a 1.8-fold increase in expression of OSF2/CBFA1 (a gene involved in commitment to the osteoblast pathway), by increased mRNA expression and protein secretion for alkaline phosphatase (ALP), type I procollagen and osteocalcin (OC) (except for OC protein), and by increased mineralized nodule formation. Transient transfection with Osf2/Cbfa1 antisense oligonucleotide substantially reduced BMP-2-stimulated expression of ALP mRNA and protein. The effects of BMP-2 on adipocyte differentiation varied: expression of peroxisome proliferator-activated receptor gamma2 (a gene involved in commitment to the adipocyte pathway) was unchanged, mRNA expression of the early differentiation marker, lipoprotein lipase, was increased, and mRNA and protein levels of the late differentiation marker, leptin, and the formation of cytoplasmic lipid droplets were decreased. Thus, by enhancing osteoblast commitment and by inhibiting late adipocyte maturation, BMP-2 acts to shunt uncommitted marrow stromal precursor cells from the adipocyte to the osteoblast differentiation pathway.     

辛伐他汀在骨髓基质干细胞向成骨细胞分化过程中的作用

目的 通过对小鼠骨髓干细胞体外培养的观察，研究辛伐他汀在骨髓基质干细胞向成骨细胞定向分化过程中的作用。方法 取雄性6周ICR小鼠股骨骨髓基质细胞进行原代和传代培养，应用组织化学及yon Kossa方法检测细胞碱性磷酸酶染色和细胞外基质矿化；在细胞培养早期加入辛伐他汀(实验组)或保持基础培养条件(对照组)，应用半定量RT-PCR方法分别检测两组Ⅰ型胶原蛋白(COL1)、碱性磷酸酶(ALP)、转录因子CBFA1和Osterix(OSX)在成骨细胞分化过程中的表达。结果 小鼠骨髓基质细胞经体外诱导后分化为具备碱性磷酸酶活性和矿化细胞外基质的成熟成骨细胞。实验组COL1、ALP和CBFA1表达在细胞培养第3，5天均高于对照组，OSX表达差异不明显。结论 辛伐他汀在成骨细胞分化过程中促进其相关基因的表达。     

Number and proliferative capacity of osteogenic stem cells are maintained during aging and in patients with osteoporosis.

Decreased bone formation is an important pathophysiological mechanism responsible for bone loss associated with aging and osteoporosis. Osteoblasts (OBs), originate from mesenchymal stem cells (MSCs) that are present in the bone marrow and form colonies (termed colony-forming units-fibroblastic [CFU-Fs]) when cultured in vitro. To examine the effect of aging and osteoporosis on the MSC population, we quantified the number of MSCs and their proliferative capacity in vitro. Fifty-one individuals were studied: 38 normal volunteers (23 young individuals [age, 22-44 years] and 15 old individuals [age, 66-74 years]) and 13 patients with osteoporosis (age, 58-83 years). Bone marrow was aspirated from iliac crest; mononuclear cells were enriched in MSCs by magnetic activated cell sorting (MACS) using STRO-1 antibody. Total CFU-F number, size distribution, cell density per CFU-F, number of alkaline phosphatase positive (ALP+) CFU-Fs, and the total ALP+ cells were determined. In addition, matrix mineralization as estimated by alizarin red S (AR-S) staining was quantified. No significant difference in colony-forming efficiency between young individuals (mean +/- SEM; 87 +/- 12 CFU-Fs/culture), old individuals (99 +/- 19 CFU-Fs/culture), and patients with osteoporosis (129 +/- 13 CFU-Fs/culture; p = 0.20) was found. Average CFU-F size and cell density per colony were similar in the three groups. Neither the percentage of ALP+ CFU-Fs (66 +/- 6%, 65 +/- 7%, and 72 +/- 4% for young individuals, old individuals, and patients with osteoporosis, respectively) nor the percentage of ALP+ cells per culture (34 +/- 5%, 40 +/- 6%, and 41 +/- 4%) differed between groups. Finally, mineralized matrix formation was similar in young individuals, old individuals, and patients with osteoporosis. Our study shows that the number and proliferative capacity of osteoprogenitor cells are maintained during aging and in patients with osteoporosis and that other mechanisms must be responsible for the defective osteoblast (OB) functions observed in these conditions.