Irradiation at 780 nm increases proliferation rate of osteoblasts independently of dexamethasone presence

BACKGROUND AND OBJECTIVES: We have previously shown that phototherapy increases cell growth and impairs protein secretion of fibroblasts. Our objective was to study the effect of phototherapy on osteoblast-like cells in culture treated with dexamethasone. STUDY DESIGN/MATERIALS AND METHODS: Rat calvaria osteoblast-like cells were previously treated or not with dexamethasone and then, they were irradiated or not with a GaAlAs diode laser (wavelength of 780 nm, 10 mW, 3 J/cm2). Adhesion, proliferation, and osteonectin synthesis were analyzed. RESULTS: Phototherapy increased the proliferation rate of cells independently of dexamethasone presence. Adhesion and osteonectin synthesis were not significantly influenced by laser and/or dexamethasone. CONCLUSIONS: Based on the conditions of this study we concluded that phototherapy acts as a proliferative stimulus on osteoblast-like cells, even under the influence of dexamethasone. Thus, we suggest that phototherapy can be of importance as co-adjuvant in bone clinical manipulation in order to accelerate bone regeneration.     

人骨髓基质细胞培养及向成骨细胞的诱导分化

目的研究人骨髓基质细胞体外培养及向成骨细胞诱导分化的实验方法。方法采用梯度离心法获得人骨髓基质细胞，细胞纯化后使用分化培养液将骨髓基质细胞向成骨细胞方向诱导分化。通过形态学观察、生化指标检测、细胞染色和矿化结节测定等方法，确定细胞的功能状态和分化程度。结果显微镜观察显示获得的人骨髓基质细胞生长状况良好，生化指标稳定；经分化培养液培养的细胞增殖速度明显减慢，生长状态平稳。细胞在分化培养过程中，上清液中碱性磷酸酶分泌量明显增加，细胞碱性磷酸酶染色明显浓染，随时间呈显著增强趋势；采用常规培养液培养的骨髓基质细胞，在汇合后不能形成明显的矿化结节。用分化培养液培养的人骨髓基质细胞，在14d时开始出现矿化结节，在21d时呈现密集的茜素红染色矿化结节。结论梯度离心法获得的人骨髓基质细胞生长情况良好，功能状态稳定；体外培养的人骨髓基质细胞在一定条件下可以向成骨细胞方向诱导分化，并具有良好的成骨细胞功能特征，可以满足进一步研究的需要。     

Qtracker体外标记兔成骨诱导分化后细胞的初步研究

目的 探讨Qtracker体外标记兔成骨诱导分化后细胞的特点及可行性.方法 抽取3个月龄健康新西兰大白兔骨髓,贴壁培养骨髓基质干细胞(BMSCs),传至第3代后向成骨细胞诱导,并做鉴定.Qtracker分别以1、2、4、8、16和32 nmol/10~6细胞的浓度标记成骨诱导分化后细胞,分别记为A、B、C、D、E、F组;未标记的细胞作为空白对照组(G组).分别利用荧光显微镜计数和流式细胞术两种方法枪测标记阳性率,台盼蓝拒染法检测标记后细胞存活率,甲基噻唑基四唑(MTT)法观察Qtracker染料对细胞增殖的影响.结果 兔BMSCs经诱导后能向成骨细胞诱导分化.经Qtracker标记后,荧光显微镜下胞浆呈绿色荧光.随着标记浓度的增加,A、B、C、D、E、F组细胞标记阳性率逐渐增高,于8 nmol/10~6细胞的浓度标记时,在荧光显微镜下计数,标记率可达到(93.58±2.08)%;通过流式细胞仪检测,其标记率为(95.24±1.31)%,经两种方法测定,D、E、F组间标记率差异均无统计学意义(P>0.05),且与其他各组比较差异均有统计学意义(P<0.05);G组各时间点标记阳性率均为0.以不同浓度标记后各组细胞存活率均在96%以上,且各组之间差异无统计学意义(P>0.05).标记Qtracker后对细胞的增殖无影响(P>0.05).结论 Qtracker可用于兔成骨诱导分化后细胞的体外标记,在浓度为8 nmol/10~6细胞下可得到最佳的标记率,且其对成骨诱导分化后细胞的增殖无明显影响.     

Dexamethasone-induced lipolysis increases the adverse effect of adipocytes on osteoblasts using cells derived from human mesenchymal stem cells

The increased bone marrow lipid deposition in steroid-associated bone loss diseases indicates that abnormalities in fat metabolism are associated with disease development. Recent studies have suggested that bone marrow adipocytes are secretory cells and that they may release substances that have an inhibitory effect on the differentiation and function of osteoblasts. We hypothesized that exposure of bone-marrow-derived adipocytes to corticosteroids exacerbates their deleterious effects on osteoblast metabolism and function. Adipocytes and osteoblasts derived from a human mesenchymal stem cell line (240 L) were co-cultured in the absence of direct cell contact with or without dexamethasone treatment. After 6 days of co-culture, osteoblasts demonstrated significantly lower levels of function based on lower mineralization, alkaline phosphatase activity and expression of osteogenic (Runx2, osteocalcin) mRNA marker. Dexamethasone treatment resulted in significantly lower levels of osteoblastic function compared with co-cultured cells without dexamethasone. Furthermore, conditioned media from dexamethasone-treated adipocytes induced a similar toxic effect and increased apoptosis involving activation of caspases 3/7 compared with conditioned media without dexamethasone treatment. Within the conditioned media, a substantial increase in the levels of leptin and two saturated fatty acids (FAs; stearate and palmitate) was observed after dexamethasone treatment. Although leptin supplementation failed to induce the inhibitory effect on osteoblasts, similar toxic results were produced with stearate and palmitate treatment, and an increase in intracellular reactive oxygen species was observed. Stearate- and palmitate-induced apoptosis was blocked by a reactive oxygen species scavenger pyrrolidine dithiocarbamate. These data show that saturated FAs secreted from adipocytes induce lipotoxic effects via mechanisms that may involve reactive oxygen species accumulation in osteoblasts. Our results suggest that inhibition of saturated FA secretion would protect osteoblasts against adipocytes in corticosteroid-associated bone loss diseases.     

血管内皮细胞对体外培养成骨细胞生物学特性的影响

目的通过血管内皮细胞与成骨细胞体外协同培养，探讨血管内皮细胞对成骨细胞生物学特性的影响。方法用12周自愿中止妊娠并捐赠的健康胚胎来源的成骨细胞和血管内皮细胞采取直接和间接协同培养的方法。倒置相差显微镜下观察细胞一般形态，细胞计数比较增殖能力，检测骨钙素和碱性磷酸酶(ALP)活性，研究成骨细胞成骨能力的改变。结果血管内皮细胞与成骨细胞体外协同培养具有良好的细胞相容性，协同培养使细胞增殖加快（P<0.05）。直接、间接协同培养ALP活性分别为（8.200±0.755）μmol/(min*106细胞)，(12.300±1.300)μmol/(min*106细胞)，较成骨细胞ALP活性(4.900±0.800)μmol/(min*106细胞)明显增高（P<0.05）。直接、间接协同培养骨钙素合成量分别为(3.8267±0.4077)μg/L、(5.1533±0.5965)μg/L，较成骨细胞(2.5083±0.5530)μg/L明显增高（P<0.05）。结论体外协同培养血管内皮细胞与成骨细胞，对成骨细胞的生物学特性具有良好的调控作用。     

High-pressure pulsatile lavage irrigation of fresh intraarticular fractures: effectiveness at removing particulate matter from bone

OBJECTIVE: To determine the effectiveness of high-pressure pulsatile lavage (HPL) versus bulb syringe (BS) irrigation in removing particulate matter from metaphyseal cancellous bone. DESIGN: Four grams of particulate graphite were placed in twenty distal femoral intraarticular osteotomies performed on New Zealand rabbit hind limbs. Two groups of ten specimens were then irrigated using either HPL or BS irrigation. A representative coronal section from each specimen was then prepared for histologic evaluation using 400x light microscopy. The number and distribution of graphite particles-present as small (less than 20 micrometers), medium (20 to 50 micrometers), and large (greater than 50 micrometers) aggregates-were then recorded. RESULTS: The mean maximum perpendicular distance of graphite aggregates of all sizes from the osteotomy site was 12.4 millimeters (+/-SD 2.5) in the HPL group and 12.5 millimeters (+/-SD 2.0) in the BS group (p > 0.5). The mean number of aggregates within four 400x fields (1.08 millimeters) of the osteotomy site was 21.9 (+/-SD 22.0) in the HPL group and 21.8 (+/-SD 27.5) in the BS group (p > 0.5). The mean total number of aggregates in the area surveyed was 129.4 (+/-SD 79.6) in the HPL group and 137.5 (+/-SD 113.6) in the BS group (p > 0.5). Separate analyses controlling for aggregate size of the specimens also revealed no significant differences between HPL and BS irrigation. CONCLUSION: HPL and BS irrigation appear equally effective in removing particulate matter from metaphyseal cancellous bone in an intraarticular fracture model. Furthermore, HPL does not appear to drive particulate matter farther into metaphyseal cancellous bone than BS irrigation.     

Over-expression of TIMP-1 in osteoblasts increases the anabolic response to PTH

Intermittent PTH treatment induces structural changes that affect cancellous bone mass and have led to its indication for the treatment of osteoporosis. PTH is also known to upregulate the expression of matrix metalloproteinases (MMP) in osteoblasts. We wanted to find out whether inhibiting osteoblastic MMPs can affect the anabolic action of PTH in vivo. We had shown previously that mice over-expressing TIMP-1 (tissue inhibitor of MMPs) specifically in osteoblasts display an increase in bone mineral density and bone mass combined with an overall decrease in bone turnover. In the present study, 10-week-old wild-type (WT) and transgenic (TG) mice were treated with PTH at 40 microg/kg/day for 1.5 months. DEXA analysis was performed before and after treatment, and histomorphometric and molecular analysis were carried out at the end of the experiment. Our findings indicate that the transgene boosted the anabolic action of PTH. The femurs of PTH-treated TG mice displayed a greater increase in bone mineral density and trabecular bone volume than treated WT mice. Interestingly, the positive effect of the transgene on the action of PTH resulted from both reduced bone resorption activity and an increase in the bone formation rate. Osteoclastic surfaces that were increased in PTH-treated WT mice remained unchanged in TG mice, suggesting a decrease in osteoclastic differentiation. Histomorphometric data also indicate that PTH administration increased osteoblast activity in TG mice and affected the number of osteoblasts in WT mice. In conclusion, we demonstrate that inhibiting osteoblastic MMPs can potentiate the anabolic effect of PTH by decreasing osteoclast activity and increasing osteoblast activity. Our data also suggest that osteoblastic MMPs have some role in mediating the anabolic effects of PTH in vivo and indicate that inhibitors of MMPs could constitute a new therapy for degenerative diseases.     

长春新碱对体外培养成骨细胞的影响

目的　观察抗癌药物长春新碱对体外培养成骨细胞的作用。方法　在新生SD大鼠头颅骨次代成骨细胞 (OB2 )培养液中分别加入不同浓度 (10 -1～ 10 -9g/L)长春新碱 ,分别观察OB2 的增殖功能 (用波长 5 70nm处OD值表示 )、分化功能 [用碱性磷酸酶 (ALP)活性表示 ]和矿化功能 (用矿化结节数 /视野表示 )。结果　OD值实验组为 0 0 91± 0 0 0 5～ 0 2 97± 0 0 44 ,对照组为 0 34 7± 0 0 35 ;ALP(U/g蛋白质 )活性实验组为 77± 12 ,对照组为 81± 4;矿化结节数 /视野 (个 )实验组为 1 0±0 816,对照组为 1 5± 1 0。结论　从绝对数来看 ,各浓度长春新碱对OB2 的增殖、分化和矿化功能均具抑制作用。与对照组比较 ,长春新碱对OB2 增殖功能的抑制作用具有极显著性意义 (P <0 0 1)。     

降钙素对体外培养成骨细胞的影响

目的观察破骨细胞抑制剂--降钙素(密钙息)对体外培养成骨细胞的作用.方法在新生SD大鼠头颅骨第二继代成骨细胞(OB\-2)培养液中分别加入不同浓度(10-4～10-12g/ml)的密钙息,分别观察OB\-2的增殖功能(用波长570nm处OD值表示),分化功能[用碱性磷酸酶(ALP)活性表示]和矿化功能(用矿化结节数量/视野表示).结果OD值(均值+标准差)为0.323±0.101～0.523±0.158;ALP活性(均数±标准差)为(0.104±0.012)U/mg蛋白质;矿化结节数量/视野(均数±标准差)为5.75±0.957个.结论与对照组比较,适当浓度(10-8～10-12)的密钙息对OB\-2的增殖,分化和矿化功能均具有促进作用,10-10g/ml浓度密钙息作用尤其显著,它们的作用显著性差异为促进OB2增殖功能P＜0.05;促进OB2分化功能P＜0.01和促进OB2矿化功能P＜0.001.     

体外培养成骨细胞的研究进展

随着体外细胞培养技术的发展,人们已经从许多动物的颅骨、骨髓基质、骨膜及骨外组织中成功培养出了具有典型成骨细胞特性的细胞,研究表明培养出的成骨细胞具有良好的生物学特性,在不同环境下可以形成骨组织,联合支架的应用,构建组织工程骨,将其植入体内修复骨缺损.现就成骨细胞的来源、分化调控因子、复合移植及中医药方面的研究进展作一综述.     

成人成骨细胞体外培养

目的 改良成人成骨细胞消化培养方法,以满足在细胞学水平进行骨代谢性疾病研究的需要。方法 取无菌骨碎片,先用胰蛋白酶消化多次,以去除血的血细胞和成纤维细胞;骨片经和培养后,再次使用胰蛋白酶消化,得到大量纯净成骨细胞。细胞长至汇合后生成黑色结节。分别采用ＮＰＰ底物法,＾１２５Ｉ标记放射免疫（ＲＩＡ）法和Ⅰ、Ⅲ型胶原顺序沉淀提、ＳＤＳ－ＰＡＧＥ还原电泳法测定细胞上清中成骨细胞主要特征性分泌物;大量碱性磷     

静磁场不同处理时间对体外培养成骨细胞增殖与分化的影响

目的:研究静磁场不同处理时间对体外培养成骨细胞增殖与分化成熟的影响。方法:原代培养大鼠颅骨成骨细胞,传代后随机分为9组,用磁场强度为3.9mT的静磁场,分别处理0(对照组)、0.5、1.0、1.5、2.0、2.5、3.0、3.5、4.0h。倒置相差显微镜下观察细胞形态;48h后检测细胞增殖情况;第3、6、9、12天测定碱性磷酸酶活性和钙盐沉积量;第8天进行碱性磷酸酶染色;第10天进行茜素红钙化结节染色;在SMFs(static magnetic fields)处理0、24、48、72h后,Real-time PCR检测骨形态发生蛋白(bone morphogenetic protein-2,BMP-2),Runx-2,骨保护素(osteoprotegerin,Opg)的mRNA表达水平变化。结果:与对照组相比,磁场组均促进细胞增殖(P<0.01或P<0.05),也能明显促进其分化成熟,表现为提高细胞的ALP活性,促进钙盐沉积量,提高BMP-2、Runx-2和Opg的mRNA表达。结论:磁感应强度为3.9mT的静磁场处理体外培养成骨细胞2.5h时,其对成骨细胞的增值与分化效果最为明显。     

妊马雌酮对大鼠体外培养成骨细胞作用的研究

目的 探讨妊马雌酮对大鼠体外培养成骨细胞的作用效果及对成骨细胞增殖的作用机理。方法 采用新生大鼠颅骨进行分离成骨细胞，并在含10％的胎牛血清DMEM中进行体外培养，分别加入高、低剂量妊马雌酮并与阴性组进行对照，用MTT法进行成骨细胞增殖和活性检测，用Western免疫印迹检测蛋白激酶C、抗凋亡蛋白Bag-1表达。结果 在培养24h后，妊马雌酮对培养成骨细胞增殖有明显的促进作用，培养到48h促进作用更为明显，表现出一定的时间相关性。妊马雌酮能明显提高体外成骨细胞PKC的表达量和抗凋亡蛋白Bag-1的表达量。结论 妊马雌酮可促进成骨细胞增殖并明显提高PKC和抗凋亡蛋白Bag-1的表达量是其防治骨质疏松的机理之一。     

葛根素对人牙周膜干细胞成骨分化的作用

目的:探讨葛根素对人牙周膜干细胞(human periodontal ligament stem cells,hPDLSCs)成骨分化的影响。方法:向体外培养的hPDLSCs中分别加入0.01mmol/L、0.1mmol/L和1mmol/L葛根素作为实验组,另设阳性对照组和空白组。MTT法检测葛根素对hPDLSCs活性影响,采用免疫荧光法、碱性磷酸酶(alkaline phosphotase,ALP)试剂盒、茜素红染色及qRT-PCR对葛根素作用后hPDLSCs生物学特性作初步观察。结果:0.01mmol/L葛根素可明显促进hPDLSCs增殖,免疫荧光染色显示实验组I型胶原蛋白(collagen-I,COL-I)和骨桥蛋白(osteopontin,OPN)均阳性表达;与空白组对比,实验组诱导7天后ALP活性升高,14天后茜素红染色见矿化结节形成,qRT-PCR检测ALP和骨钙蛋白(osteocalcin,OCN)分别在加药后7天和14天表达上调。结论:葛根素能够促进牙周膜干细胞向成骨细胞分化。     

Hypoxia increases insulinlike growth factor gene expression in rat osteoblasts

Vascular disruption secondary to fracture leads to a hypoxic zone of injury where the oxygen tension at the center of the wound is quite low. In this dynamic microenvironment, a number of growth factors are elaborated to stimulate the synthetic processes of fracture repair. Previously the authors have shown the hypoxia-induced increase of vascular endothelial growth factor expression in osteoblasts. The purpose of these experiments was to examine osteoblast expression of insulinlike growth factors (IGF) I and II--cytokines believed to play a role in increased collagen synthesis, chemotaxis, and proliferation of osteoblasts in response to hypoxia. Primary cell cultures of osteoblasts isolated from neonatal rat calvaria were subjected to hypoxia (PO2 = 35 mmHg) for 0, 3, 6, 24, and 48 hours. Northern blot analysis of ribonucleic acid (RNA) from resulting cultures demonstrated a more than 60% increase in IGF-II messenger RNA (mRNA) expression after 3 hours of hypoxia. IGF-II mRNA expression continued to increase through later time points to 200% and 260% of baseline at 24 and 48 hours respectively. In contrast, IGF-I demonstrated no significant change in mRNA expression compared with baseline control (normoxia) cultures. In these experiments the authors have demonstrated a hypoxia-induced increase in IGF-II but not IGF-I in primary osteoblasts. The differential expression of these two growth factors may underscore important differences in the behavior of osteoblasts in the hypoxic fracture microenvironment. Taken together, these data add additional support to the theory that hypoxia induces gene-specific changes in expression of molecules important to extracellular matrix formation for successful bone healing.     

尼莫地平(nimodipine)对体外培养成骨细胞的影响

目的观察钙通道阻断剂--尼莫地平(nimodipine)对体外培养成骨细胞的作用.方法在新生SD大鼠头颅骨第二继代成骨细胞(OB2)培养液中分别加入不同浓度(10-4～10-12g/ml)尼莫地平,分别观察OB2的增殖功能(用波长570nm处OD值表示),分化功能[用碱性磷酸酶(ALP)活性表示]和矿化功能(用矿化结节数量/视野表示).结果OD值(均值±sf标准差)为0.121±0.009～0.411±0.035;ALP活性(均数±标准差)为0.092±0.008U/mg蛋白质;矿化结节数量/视野(均数±标准差)为1.250±0.893个.结论与对照组比较,尼莫地平各浓度对OB2的增殖、分化和矿化功能作用各异.当尼莫地平浓度为10-9g/ml时,对OB2的增殖和分化功能具有刺激作用(p＜0.05),对OB2的增殖功能具有明显的抑制作用(p＜0.01);当浓度降低(10-11g/mi)时,对OB2的增殖功能失去作用(p＞0.05).     

Heparin facilities proliferation of human osteoblasts in vitro

It is a well-known fact that long-term application of heparin can lead to osteoporosis. To learn more about the mechanisms of heparin-induced osteoporosis, we exposed human osteoblasts in vitro to heparin in various concentrations. We found an increased proliferation rate, especially in concentrations used therapeutically in humans (0.1-0.2 IU/ml). In our experiments fetal calf serum (FCS) was able to heighten the positive effect of heparin, showing a synergism between heparin and FCS.