地塞米松在人骨髓基质细胞诱导成骨细胞中的作用

目的 通过对人骨髓基质细胞体外培养及检测，研究地塞米松在骨髓基质细胞体外增生并向成骨细胞定向分化过程中的应用。方 法骨折内固定术扩髓前收集髓腔内骨髓进行原代和传代培养，传代后改用条件培养基（含地塞米松）和基础培养基（不含地塞米松）分别进行培养，应用组织化学及von Kossa方法检测细胞碱性磷酸酶和细胞外基质矿化程度；半定量RT-PCR方法检测Cbfal mRNA和Osterix mRNA在细胞培养过程中不同时间点的表达。并观测地塞米松对上述成骨细胞相关基因表达的影响。结果 条件培养基组细胞Cbfal mRNA和Osterix mRNA的表达峰值高于基础培养基组细胞，并且峰值出现的时间提前。结论 地塞米松通过促进Cbfal mRNA和Osterix mRNA的表达而促进骨髓基质细胞向成骨细胞分化。     

In vitro effects of chemotherapeutic agents on human osteoblast-like cells

Osteopenia is a complicating problem that may occur during and after treatment for childhood malignancy. Clinical studies suggest that chemotherapeutic agents directly affect osteoblasts in vivo. Since combinations of agents are used for treatment, we individually investigated the chemosensitivity of human osteoblast-like cells to 11 of the chemotherapeutic agents used. The relative chemosensitivity of osteoblast-like cells representing different stages of cell differentiation was also examined. Cell numbers were evaluated following culture of an established human osteoblast-like cell line (MG63) for 3 days with clinically relevant concentrations of the chemotherapeutic agents. The chemosensitivity of MG63 cells was compared to that of a human osteoprogenitor cell line (HCC1) and primary osteoblast-like (HOB) cells derived from pediatric bone. Cell numbers were reduced by all agents in all cell types, although there was a varied response between agents at equimolar concentrations. In MG63 cells the lowest concentration of agent significantly reducing cell numbers varied between agents, for example, methotrexate (10(-7) M), vincristine (10(-9) M), and etoposide (10(-7) M) (all P <0.01). The less differentiated osteoblast phenotypes were significantly more chemosensitive at equimolar concentrations of methotrexate, vincristine, asparaginase, and dexamethasone than more differentiated phenotypes (all P <0.01). Furthermore, four agents significantly increased alkaline phosphatase (AP) activity in HOB cells. We conclude that individual chemotherapeutic agents added to osteoblast cell cultures reduce cell numbers, with osteoblast precursor cells being preferentially depleted. These results suggest that most of the agents may contribute to osteopenia in childhood malignancy by direct effects on cell numbers.     

In vitro culture of human autologous osteoblast cells on natural bone mineral

Different methods are available for the treatment of osseous defects. In recent years the use of autologous bone was established as the golden standard. However, significant disadvantages are limited availability of the bone graft and its harvest implies additional morbidity for the patient. Alternatives to the use of autologous bone, as allogeneic bone from bone banks or biomaterials like hydroxyapatite are therefore of special interest. However, the currently available methods have severe disadvantages; allogenic bone carries a high risk of transmitting infectious diseases, most biomaterials show an unsatisfying osseous integration as well as prolonged healing with disability for the patient. Therefore, the aim has to be the development of a biomaterial that is as close as possible to human bone. In this in vitro study the natural bone mineral Bio-Oss/Orthos was used as a matrix for human osteoblast-like cells isolated from bone marrow of healthy patients. Even after three months the cell showed typical osteblast-like behaviour. Histologic evaluation demonstrated the ability of Bio-Oss/Orthos to guide cell growth within its matrix structure and therefore mimics in vivo situation of the healthy bone. The results show that culturing human osteoblast-like cells under standardised conditions is possible and that the combination of human osteoblast-like cell with an appropriate matrix may have the potential for a new treatment option of osseous defects.     

Expression of fibrillins and other microfibril-associated proteins in human bone and osteoblast-like cells

Fibrillin-containing microfibrils are structural components of extracellular matrices of a diverse range of tissues, including bone. Their importance in bone biology is illustrated by the skeletal abnormalities manifest in the congenital disorder, Marfan syndrome, which results from mutations in the fibrillin-1 gene. We investigated the expression of fibrillins and other microfibril-associated proteins in human bone and bone-derived osteoblasts. Analysis of RNA extracted from cancellous bone showed expression of mRNAs encoding fibrillin-1 and -2, MAGP-1 and -2, LTBP-2, and MP78/70 (Big-h3). In demineralized normal mature bone, fibrillin-1 was immunolocalized to fibrils within the bone matrix and pericellularly to cells lining the endosteal surfaces of trabecular bone, some osteocytes, and cells associated with blood vessels. LTBP-2 was also identified at the endosteal surface and within the bone matrix in a lamellar fashion. In addition, primary osteoblast-like cells cultured from human trabecular bone (obtained from patients at joint replacement surgery) were found to express abundant mRNA for fibrillins and associated glycoproteins. Moreover, using western blot analysis, fibrillin-1 protein was shown to be secreted into the medium and to be deposited into the cell layer. Immunofluorescence staining of the cell layer visualized fibrillin-1 in the matrix as a three-dimensional network of fine filaments. Expression of fibrillin-1 by osteoblast-like cells was constitutive, and a number of skeletally active agents had little effect on mRNA or protein levels. These results show that human osteoblasts from mature bone express fibrillins and other microfibril-associated proteins, and suggest a role for these molecules in adult human bone.     

Human bone bank allografts stimulate bone resorption and inhibit proliferation in cultures of human osteoblast-like cells

Incorporation of a frozen human bone allograft requires osteoclast activity and ingrowth of recipient osteoblast precursors. We examined the effects of allografts on human osteoblasts. Allografts stimulated a release of factors from normal human osteoblast-like cells, capable of inducing osteoclastic bone resorption in vivo. Further allografts inhibited osteoblast proliferation in cultures. the response was detectable within 4 days of culture and was still present after 3 weeks. Devitalized bone autografts had a similar effect. This suggests that bone bank grafts may induce a resorptive reaction at the recipient site by stimulating release of factors from osteoblasts capable of inducing osteoclastic resorption. the storage temperature was crucial for preservation of the response, since the activity was lower in allografts stored for 6 months at -20°C than in those stored at -80°C.     

Cigarette smoke extract inhibits chemotaxis and collagen gel contraction mediated by human bone marrow osteoprogenitor cells and osteoblast-like cells

Cell migration and matrix remodeling are key events in tissue repair and restructuring. Osteoblasts are responsible for the production of new bone matrix during bone remodeling. The activity of these cells can be modulated by a number of factors. The current study evaluated the hypothesis that cigarette smoke extract can alter repair and remodeling responses of human osteoprogenitor cells and osteoblast-like cells and, therefore, could explain one mechanism by which cigarette smoking leads to osteoporosis. Human osteoprogenitor cells were isolated from normal human bone marrow and maintained in culture under either control conditions or conditions that induced differentiation into osteoblast-like cells. Both cell types migrated toward fibronectin and PDGF-BB as chemoattractants. Neither responded to TGF-beta1. The osteoprogenitor cells were more active in their chemotactic response. The chemotactic response of both cell types was inhibited by cigarette smoke extract in a concentration-dependent manner. Both cell types, when cultured in three-dimensional native collagen gels maintained in floating culture, induced contraction of their surrounding matrices. Contraction was augmented by serum, PDGF-BB, and TGF-beta1. Osteoprogenitor cells were less active in inducing contraction than were osteoblast-like cells. Contraction of both cell types was inhibited by cigarette smoke extract. Cigarette smoke extract also inhibited the production of fibronectin by both cell types maintained in three-dimensional culture. Addition of exogenous fibronectin partially restored the ability of the cells to contract three-dimensional collagen gels. The current study demonstrates that cigarette smoke can interfere with the ability of bone cells to participate in repair and remodeling events. Such an effect may be one mechanism leading to the development of osteoporosis.     

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.     

Stimulation of human prostate cancer cell lines by factors present in human osteoblast-like cells but not in bone marrow

Secondary deposits of prostate tumours are frequently found in the skeleton where they produce osteoblastic lesions. In this study both osteoblast-like cells and bone marrow from the proximal femur have been cultured to determine whether or not they can release factors which could support the growth of secondary prostate tumours. Media conditioned by both osteoblast-like cells (OBCM) and bone marrow were examined for their potential to stimulate prostate carcinoma cell lines. Whilst the results obtained demonstrated that OBCM could enhance the growth of both the hormone sensitive (LNCaP) and hormone unresponsive (PC-3 and DU-145) prostate carcinoma cell lines, no proliferative effect could be shown on cell lines derived from cancers of the breast, bladder, and liver. Significantly, media conditioned by either bone marrow or human skin fibroblasts also had no effect on the growth of prostate carcinoma cell lines. This study supports the possibility that the proliferation of prostate cancer cells at secondary skeletal sites, in vivo, may be due to osteoblast derived factors. © 1995 Wiley-Liss, Inc.     

蛇床子素对模拟微重力引起的骨质流失的防治作用

目的研究蛇床子素对模拟微重力导致大鼠骨质流失的防治作用。方法将Wistar大鼠随机分为3组:对照组(Ctrl)、尾吊组(HLS)、尾吊给药组(OST)。4周后取后肢骨用双能骨密度仪及万能试验机分别测量各组股骨骨密度及生物力学,并通过micro CT分析骨小梁参数;用Van Gieson(VG)染色观察胫骨形态变化。通过qRT-PCR检测胫骨骨保护素(OPG)和核因子-κB受体激活剂配体(RANKL)mRNA表达水平。用ELISA试剂盒检测血清中骨特异性碱性磷酸酶(BALP)、骨钙素(OCN)和抗酒石酸酸性磷酸酶-5b(TRACP-5b)的含量变化。结果与对照组相比,尾吊组大鼠股骨骨密度及生物力学参数显著降低(P0.05),与尾吊组相比,尾吊给药组骨密度及生物力学显著上升(P0.05);尾吊组骨小梁体积分数(BV/TV)、厚度(Tb.Th)显著降低(P0.01),间距(Tb.Sp)显著升高(P0.01),尾吊给药后BV/TV、Tb.Th及Tb.Sp显著降低(P0.01);VG染色观察尾吊组胫骨骨小梁间隙变大,数量减少,而尾吊给药组骨小梁骨髓腔减少,数量增多;尾吊组血清BALP和OCN浓度显著降低(P0.05),TRACP-5b浓度显著升高(P0.05),当给予蛇床子素后,BALP浓度显著升高(P0.05),而TRACP-5b浓度显著降低(P0.05);尾吊组胫骨OPG/RANKL比值显著降低(P0.05),尾吊给药后,OPG/RANKL比值显著升高(P0.05)。结论蛇床子素可通过促进骨形成和抑制骨吸收两方面有效防治模拟微重力导致的骨质流失。     

Ascorbate uptake by ROS 17/2.8 osteoblast-like cells: substrate specificity and sensitivity to transport inhibitors

Ascorbate (reduced vitamin C) is required for bone formation. We have shown previously that both the osteoblast-like cell line ROS 17/2.8 and primary cultures of rat calvarial cells possess a saturable, Na(+)-dependent uptake system for L-ascorbate (J Membr Biol 111:83-91, 1989). The purpose of the present study was to investigate the specificity of this transport system for organic anions and its sensitivity to transport inhibitors. Initial rates of ascorbate uptake were measured by incubating ROS 17/2.8 cells with [L-14C]ascorbate at 37 degrees C. Uptake of [L-14C]ascorbate (5 microM) was inhibited 98 +/- 1% by coincubation with unlabeled L-ascorbate (3 mM) and 48 +/- 4% by salicylate (3 mM), but it was not affected by 3 mM formate, lactate, pyruvate, gluconate, oxalate, malonate, or succinate. Uptake of the radiolabeled vitamin also was not affected by acute (1 minute) exposure of the cells to the Na+ transport inhibitors amiloride and ouabain or the glucose transport inhibitor cytochalasin B. In contrast, anion transport inhibitors rapidly (less than 1 minute) and reversibly blocked [L-14C]ascorbate uptake. In order of potency, these drugs were 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) approximately equal to sulfinpyrazone greater than furosemide approximately equal to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). These findings indicate that the ascorbate transporter is relatively specific for the ascorbate anion, since other organic anions (with the exception of salicylate) did not compete with ascorbate for uptake. Rapid and reversible inhibition by the impermeant antagonists DIDS and SITS suggests that they interact directly with the ascorbate transporter, consistent with location of the transport system in the plasma membrane.     

Three isolation techniques for primary culture of human osteoblast-like cells: a comparison

The culture of osteoblast-like cells of human origin has become an important experimental model in bone biology. We report here a comparison and evaluation of three of the most widely used systems available today: bone marrow stroma cell cultures (BMSC), human osteoblast explant cultures (hOB) and osteoblast explant cultures from collagenase-treated bone (hOBcol). Cultures from 16 bone specimens obtained from various donors were established and their expression of the osteoblast phenotype were then compared in secondary cultures by use of biochemical markers. BMSC had the highest basal and 1,25-dihydroxyvitaminD3 (1,25(OH)2D3)-induced alkaline phosphatase activities in all cell isolations, with levels approximately twice those in explant cultures. Basal osteocalcin secretion was low-to-undetectable in all cell cultures but was detected in 1,25(OH)2D3-stimulated cultures. BMSC produced half of the amount of osteocalcin synthesized in explant cultures. The BMSC cultures also synthesized the lowest amounts of type I collagen, whereas collagen type III synthesis did not differ significantly among the various cultures. When secondary cultures were treated with 100 nM dexamethasone in the presence of ascorbic acid (50 microg/mL) and beta-glycerophosphate (10 mM), cultures deposited calcium mineral into the cell layer within 2-4 weeks. PTH-induced cAMP formation was detected in only 5 of 15 isolations and no consistent isolation-dependent response pattern was seen. We conclude that BMSC cultures differ significantly from explant cultures obtained from the same bone specimen. However, all cultures represent cells which can differentiate further and induce mineralization of the extracellular matrix in response to osteoinductive drugs.     

The roles of TNFR1 in lipopolysaccharide‐induced bone loss: Dual effects of TNFR1 on bone metabolism via osteoclastogenesis and osteoblast survival

LPS (lipopolysaccharide), a major constituent of Gram‐negative bacteria, regulates proliferation and differentiation of osteoclasts directly or indirectly. This study sought to investigate the functions of the RANK/RANKL pathway in LPS‐induced bone loss in vivo. Wild‐type mice or TNFR1?/? mice were injected LPS with or without osteoprotegerin (OPG) and analyzed histologically. Bone volume was reduced by LPS injection in all groups, and OPG administration prevented the LPS‐induced bone loss regardless of genotypes. LPS‐induced enhancement of osteoclastogenesis in wild‐type mice was blocked by OPG administration. LPS or OPG did not affect osteoclastogenesis in TNFR1?/? mice. Interestingly, osteoblast surface was remarkably reduced in LPS‐treated TNFR1?/? mice as a result of enhanced osteoblast apoptosis. TRAIL, induced by TNF‐α in BMC, triggered apoptosis of primary osteoblast only when TNFR1 signal was ablated in vitro. In conclusion, RANK signaling plays a prominent role in osteoclastogenesis downstream of LPS. Furthermore, TNFR1 regulates bone metabolism through not only the regulation of osteoclast differentiation but also osteoblast survival. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:657–663, 2010     

Apoptosis induced by photosensitizers (Perylquinone derivatives) in human carcinoma cells: a possible relevance to photodynamic therapy

Novel photosensitizers Hypocrellin A (HA) and Hypocrellin B (HB), lipid soluble perylquinone derivatives of the genus Hypericum have a strong photodynamic effect on tumors and viruses. However, the mechanisms of tumor cell death induced by HA and HB are still unclear. In this study, we attempt to elucidate the photodynamic effects of HA and HB compounds in poorly differentiated (CNE2) and moderately differentiated (TW0-1) human nasopharyngeal carcinoma (NPC) cells as well as human mucosal colon (CCL-220.1) and bladder (SD) cells. Using these cell lines we investigated few hall marks of apoptotic commitments in a drug and light dose dependent manner.Tumor cells photoactivated with HA and HB showed cell size shrinkage and an increase in the sub-diploid DNA content. A loss of membrane phospholipid asymmetry associated with apoptosis was induced by all tumor cell lines as evidenced by the externalization of phosphatidylserine. Western blot analysis of poly (ADP-ribose) polymerase, a caspases substrate, showed the classical cleavage pattern (116 to 85kDa) associated with apoptosis in HA and HB-treated cell lysates. In addition, PARP cleavage was blocked by using tetrapepdide caspases inhibitors such as DEVD or z-VAD. These results demonstrate that tumor cell death induced by HB and HA is mediated by caspase proteases. This study also identifies both colon and bladder cells were more sensitive cell lines than NPC (CNE2 and TWO-1) cell lines.     

Leptin: a potential mediator for protective effects of fat mass on bone tissue

Body weight is among the most powerful predictors of bone status, and adipose tissue plays a substantial role in weight-related protective effects on bone. An understanding of the mechanisms underlying the relation between adipose tissue and bone may open up new perspectives for treatment. Leptin, which is known to regulate appetite and energy expenditures, may also contribute to mediate the effects of fat mass on bone. Although reported data are somewhat conflicting, there is some evidence that leptin may decrease bone formation via a central nervous effect and may stimulate both bone formation and bone resorption via direct peripheral effects on stromal precursor cells. The net result of these central and peripheral effects may depend on serum leptin levels and blood-brain barrier permeability, of which the first increase and the second decrease as obesity develops. Further work is needed to improve our understanding of these effects.     

胰岛素和胰岛素样生长因子对人成骨样细胞MG63骨钙素基因表达的影响

目的在一定条件下对人成骨样细胞MG63进行体外培养,并用胰岛素、胰岛素样生长因子-1（Insulin like growth factor-1,IGF-1）进行干预,以17-β雌二醇做阳性对照。观察药物干预对MG63细胞骨钙素基因表达的影响。为糖尿病性骨质疏松的治疗提供理论和实验依据。方法用DMEM培养基培养MG63细胞,并以胰岛素、IGF-1、雌二醇进行干预。抽提各样本总RNA后,行逆转录并对骨钙素的cDNA进行荧光扩增和定量。结果各药物组内基因拷贝数差异均有统计学意义（P〈0.05）,各组药效呈浓度依赖性增加趋势。在本实验所选的药物浓度范围内,各药物组的最高药效浓度均为所用的最高药物浓度,分别为200nmol/L、100nmol/L、1000nmol/L。3组药物的骨钙素拷贝数增长率比较差异有统计学意义（P〈0.05）,胰岛素组的拷贝数增长率明显高于雌二醇组和IGF1组（P〈0.05）,IGF-1与雌二醇组拷贝数增长率比较差异无统计学意义（P〉0.05）。结论胰岛素、IGF-1和雌二醇一样,均有显著促进MG63分化的作用。     

G-B-R复配物对模拟微重力效应诱导骨丢失的防护作用

目的 从天然产物中寻找到对微重力效应诱导骨丢失具有防护作用的复配物。方法 通过24种天然产物对骨髓干细胞和成骨细胞的增殖活性,筛选出具有较强效果的天然产物并对其进行复配;随后通过复配物对模拟微重力效应下昆明小鼠骨质丢失的影响来评价其骨丢失防护作用。结果银杏叶和荞麦花粉的复配物显示出最强的骨髓干细胞增殖活性;熟地黄显示出最强的成骨细胞增殖活性;在模拟微重力环境下不同剂量的银杏叶、荞麦花粉与熟地黄的复配物均显示出不同程度的骨丢失防护作用;高剂量组可以显著提高模型组小鼠血清中碱性磷酸酶的活性及股骨中的有机物含量。结论 通过对天然产物促骨髓干细胞和成骨细胞增殖活性进行筛选和复配,发现所获得的G-B-R复配物对微重力效应诱导的骨丢失具有良好的防护作用。     

Modification of gene expression induced in human osteogenic and osteosarcoma cells by culture on a biphasic calcium phosphate bone substitute

Bone hybrids made of bioceramics seeded with mesenchymal or osteoblastic cells are very promising alternatives to autologous bone graft. Along this line, the development of in vitro models, dedicated to analyze the influence of these biomaterials on osteogenic cells, will help to improve the performance of these bone substitutes. In the present work we analyzed the effects of a macroporous biphasic calcium phosphate ceramic (BCP, Triosite) on three different human osteosarcoma cell lines and on human primary osteogenic cells and compared this culture substratum to traditional culture on plastic. We showed that all these osteoblastic cells adhere and proliferate on the trabecular BCP blocks, with a different spatial organization for osteosarcoma cells compared to normal osteogenic cells. We also demonstrated that osteoblastic marker genes such as Cbfa1, type I collagen, osteonectin, osteopontin, and osteocalcin were expressed at similar levels by these cells cultured on either substratum, suggesting that adhesion to BCP does maintain the osteoblastic phenotype of these cells. Next, we provided the first evidence of differences of cytokine expression profiles revealed on this Ca-P ceramic as compared to expression in classical culture. These modifications affected the expression of cytokines such as TGF-β1, G-CSF, and IL-3 and were quantitatively different between osteosarcoma cells and normal osteogenic cells. Given the role of these cytokines in bone biology and in hematopoiesis, these results obtained in vitro suggest that the BCP ceramic studied here could stimulate osteogenesis in vivo by activating cellular processes during bone formation and healing. This study highlights the notion that the nature of the culture substratum must be taken into account when studying bone cell biology in vitro. Owing to the nature and spatial organization of the BCP, our hypothesis is that culture on BCP is closer to the physiological situation than culture on plastic.